Peptide and polypeptide inhibitors of complement C1s

ABSTRACT

The complement system plays an important role in providing resistance to infections and in the pathogenesis of tissue injury. Yet an inappropriate activation of complement can result in a variety of disorders. The present invention provides C1s catalytic site-directed moieties, C1s exosite binding moieties, and bivalent polypeptide inhibitors comprising such moieties, which can be used to treat conditions characterized by inappropriate complement activation.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisionalapplication No. 60/212,998 (filed Jun. 21, 2000), the contents of whichare incorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates generally to new peptides andpolypeptides, which inhibit complement C1s.

BACKGROUND OF THE INVENTION

[0003] The complement system is considered an ancient part of the immunesystem, which serves to discriminate self and non-self (see, forexample, Rother et al. (Eds.), The Complement System, Second Edition(Springer-Verlag 1998), Morely and Walport, The Complement Factsbook(Academic Press 1999), and Morgan (Ed.), Complement Methods andProtocols (Humana Press, Inc. 2000)). Although the complement systemplays an important role in providing resistance to infections, aninappropriate activation of complement can result in a variety ofdisorders.

[0004] There are two main pathways for complement activation, which areknown as the classical and alternative pathways. Both pathways comprisea cascade of enzyme activation, which leads to the production of aterminal membrane attack complex that targets immune complexes ormicroorganisms. The alternative pathway is activated by the chancebinding of C3b with the surface of a microorganism. The classicalpathway is the principal antibody-directed mechanism for the activationof complement. C1, the first enzyme complex in the classical pathway, isa pentamolecular complex consisting of a single C1q molecule, and twoC1r and C1s molecules. In the classical pathway, an antibody binds withC1q, which causes the activation of the C1r molecules. These activatedproteins then cleave the C1s molecules to form active C1s serineproteases, which act on the next two components of the classicalcomplement pathway, C4 and C2. Cleaved portions of these complementproteins, known as C4b and C2a, then form C3 convertase, which goes onto cleave the next component in the cascade, C3. Thus, C1s plays a keyrole, because one C1s molecule can generate multiple C4b molecules,which have an amplification effect on the system.

[0005] Molecules that inhibit complement may be beneficial for treatmentof diseases in which complement activation has been shown to occur, suchas adult respiratory distress syndrome, ischemia-reperfusion injury(myocardial infarct, stroke, skeletal muscle, lung inflammation),hyperacute rejection (transplantation), sepsis, cardiopulmonary bypass,burns, wound healing, asthma, restenosis, multiple organ dysfunctionsyndrome, trauma, hemorrhagic shock, Guillain-Barre syndrome, paroxysmalnocturnal hemoglobinuria, glomerulonephritis, systemic lupuserythematosus, rheumatoid arthritis, infertility, Alzheimer's disease,organ rejection, myasthenia gravis, multiple sclerosis, plateletstorage, serum sickness, various hemolytic anemias, and hemodialysisSee, for example, Vogt, Trends Pharm. Sci. 6:114 (1985), and Makrides,Pharm. Rev. 50:59 (1998).

[0006] Many different types of compounds have been found to beinhibitors of classical complement, including diamines, amino acids andtheir derivatives, polynucleotides, polyanions, pyridiniumsulphonylfluorides and phenothiazines (see, for example, Ashghar,Pharmac. Rev. 36:223 (1984)). Peptide inhibitors are exemplified byamino acid sequences that mimic the C1 fixing sequences of IgG,glutathione, and leupeptin (see, for example, Boackle et al., Nature282:742 (1979); Takada et al., Immunology 34:509 (1979)). A tripeptidebased on C-terminal sequences of C3a and C5a has been shown to be asubstrate for C4b2a, CVFBb and C1s, while substrate-like inhibitors ofC3 convertase have also been prepared (see, for example, Andreatta etal., In Enzyme Inhibitors, Brodbeck (Ed.), pages 261-272 (1981);Caporale et al., J. Immun. 126:1963 (1981)).

[0007] Few compounds have been found to inhibit the alternative pathway.Complestatin, a microbial product believed to bind to factor B is oneexample of such an inhibitory compound (Kaneko et al., J. Immun.124:1194 (1980)). Many inhibitors described above require relativelyhigh concentrations, and lack specificity.

[0008] Protein inhibitors of complement have been described morerecently, and include: soluble complement receptor (sCRI), a humanizedmonoclonal antibody to C5, and BD001, a recently described proteinderived from a leech, which inhibits C1s (Liszewski and Atkinson, ExpOpin, Invest. Drugs 7:323 (1998); Seale and Finney, InternationalPublication No. WO99/36439). Seale and Finney reported that BD001 hasthe following amino acid sequence: AKKKLPKCQK QEDCGSWDLK CNNVTKKCECRNQVCGRGCP KERYQRDKYG (SEQ ID NO:1). CRKCLCKGCD GFKCRLGCTY GFKTDKKGCEAFCTCNTKET ACVNIWCTDP YKCNPESGRC EDPNEEYEYD YE

[0009] The discovery of new C1s-inhibitory peptides and polypeptidesfulfills a need in the art by providing new compositions useful indiagnosis and therapy. The present invention provides such polypeptidesfor these and other uses that should be apparent to those skilled in theart from the teachings herein.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides novel peptides and polypeptidesthat can inhibit the complement system. The present invention alsoprovides methods of producing these peptides and polypeptides.

DESCRIPTION OF THE INVENTION

[0011] 1. Overview

[0012] BD001, a leech protein that inhibits C1s and factor XIIactivation, was produced in two separate expression systems: baculovirusand Pichia methanolica. The baculovirus system produced a protein thatis virtually identical to the native inhibitory protein isolated fromthe salivary complexes of the leech. That is, mass spectroscopy andN-terminal sequencing indicated that the baculorivus-derived materialhad the correct stop and start sites, N-glycosylation at the expectedposition, and tyrosine sulfation at the C-terminus on three tyrosineresidues. Moreover, bioassays of this expressed material in an isolatedenzymatic assay of C1s showed equivalence with the native leechmaterial.

[0013] In contrast, the protein expressed in Pichia methanolica lackedthe tyrosine sulfation at the C-terminus, and N-glycosylation. Inaddition, a portion of Pichia-produced BD001 molecules were truncated atthe C-terminus. This Pichia material was found to be about 10 fold lessactive in bioassays, compared with the baculorivus-expressed material.Computer modeling of C1s and other serine proteases indicated thatalterations in the C-terminus of BD001 could lead to diminished bindingwith the exosite region of C1s. This result is consistent with thedecreased activity of BDOO that contained variations in the C-terminus.

[0014] Further sequence analyses revealed that complement protein C4,which is the endogenous substrate for C1s, includes a region that has astring of anionic residues with three tyrosine residues in a similararrangement to the amino acid sequence of BD001. In addition, studieshave shown that these three tyrosine residues of C4 must be sulfated inorder for this molecule to have activity (Hortin et al., J. Biol. Chem.261:1786 (1986); Hortin et al., Proc. Nat'l Acad. Sci. USA. 86:1338(1989)). Studies described herein substantiate the importance oftyrosine sulfation for BD001 activity.

[0015] Without being bound by theory, these collective observationsindicate that the C-terminal portion of BD001 mimics a portion of C4 toconfer specificity of binding of BD001 for C1s. Accordingly, smallpeptide or peptidomimetic inhibitors of C1s can be devised, which arebased upon the BD001 sequence.

[0016] As described herein, the present invention provides complementC1s inhibitors useful as therapeutic agents. These inhibitory peptidesand polypeptides are also useful as preservatives in blood samples. Inaddition, peptides and polypeptides described herein can be used inaffinity purification procedures to isolate C1s.

[0017] In particular, the present invention provides polypeptides thatinhibit complement C1s, wherein the polypeptides are characterized bythe formula:“P-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”where amino acid residues in square brackets indicate acceptable aminoacids, numbers in parentheses indicate the number of amino acidresidues, “X₁” represents Phe-(p-CH₂)SO₃H, “X₂” represents sulfatedtyrosine, and “X₃” represents 2-sulfotyrosine (SEQ ID NO: 127). Suitablepolypeptides include polypeptides characterized by the formula:“P-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE]” (SEQ IDNO:129), the formula: “P-N-E-E-[YX₁X₂X₃]-E-[YX₁X₂X₃]-E-[YX₁X₂X₃]-E” (SEQID NO:130), or by the amino acid sequence: “PNEEY EYEYE” (SEQ IDNO:125).

[0018] The present invention also proves polypeptides that inhibitcomplement C1s, wherein the polypeptide comprise an amino acid sequencethat is characterized by the formula:“[AP]-N-[DE](2)-[X₁X₂X₃]-[DE](2)-[X₁X₂X₃]-[DE]-[X₁X₂X₃]-[DE](1,2),”where amino acid residues in square brackets indicate acceptable aminoacids, numbers in parentheses indicate the number of amino acidresidues, “X₁” represents Phe-(p-CH₂)SO₃H, “X₂” represents sulfatedtyrosine, and “X₃” represents 2-sulfotyrosine (SEQ ID NO: 126).Additional examples of complement C1s inhibitors include a peptide orpolypeptide that inhibits complement C1s, wherein the peptide orpolypeptide comprises the amino acid sequence “CRLGC” (amino acidresidues 64 to 68 of SEQ ID NO:1), and wherein the peptide orpolypeptide consists of five to thirty amino acid residues. For example,a suitable polypeptide consists of the amino acid sequence: “GCDGFKCRLGCTYGFKTDKK GCEAFCTCNT” (SEQ ID NO:53), whereas a suitable peptideconsists of the amino acid sequence: “CRLGC.” The present inventionfurther includes complement C1s inhibitors, wherein the inhibitorsconsist of:

[0019] (a) a C1s catalytic site-directed moiety (CCSDM), which isselected from the group consisting of: (i)CH₃-Lys(Cbo)-Gly-Arg-pNA-AcOH, where “Cbo” represents benzyloxycarbonyl;(ii) CH₃-Lys(Cbo)-Gly-Arg; (iii) H-D-Val-Ser-Arg-pNA.HCl; (iv)H-D-Val-Ser-Arg; (v) Leu-Xaa-Arg, where “Xaa” represents alanine,glutamine, or glycine; (vi) LQRALEILPN RVTIKANRPF LVFI (SEQ ID NO:118),

[0020]  (vii) serine protease inhibitor; (viii) heterocyclic proteaseinhibitor; (ix) transition state analogue; (x) benzamidine; (xi)X-C1-C2-A-Y, where C1 is a derivative of Arg, Lys, or Orn, characterizedby a reduced carboxylate moiety or a carboxylate moiety that isdisplaced from the α-carbon by a chemical structure characterized by abackbone chain of from 1 to 10 atoms, C2 is a non-cleavable bond, “X” ishydrogen or a continuation of the peptide backbone, “A” is a backbonechain, and “Y” is a bond; (xii) (xii) CDGFK CRLGC TYGFK TDKKG CEAFC TCNT(SEQ ID NO:121);

[0021]  and (xiii) (xiii) X-C-X(8-12)-L-Q-R,

[0022]  where “X” represents glycine, serine, or threonine, and numbersin parentheses indicate the number of amino acid residues (SEQ ID NO:140);

[0023] (b) a linker moiety that is either characterized by a backbonechain having a calculated length of between 14 Å and 20 Å, or that is apolypeptide, which has the amino acid sequence of KETAC VNIWC TDPYKCNPES GRCED (SEQ ID NO:123);

[0024]  and

[0025] (c) a C1s exosite binding moiety (CEBM), which is selected fromthe group consisting of: (i) a polypeptide characterized by the formula:“[AP]-N-[DE] (2)-[YX₁X₂X₃] -[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),” where amino acid residues insquare brackets indicate acceptable amino acids, numbers in parenthesesindicate the number of amino acid residues, “X₁” representsPhe-(p-CH2)S_(O) ₃H, “X₂” represents sulfated tyrosine, and “X₃”represents 2-sulfotyrosine (SEQ ID NO: 126); and (ii) (ii) NEDYEDYEYD(SEQ ID NO:119);

[0026] wherein the C1s catalytic site-directed moiety is bound to thelinker moiety, the linker moiety is bound to the C1s exosite bindingmoiety.

[0027] Suitable CCSDM moieties include serine protease inhibitorsselected from the group consisting of phenylmethylsulfonylfluoride,diisopropylflouorophosphate, tosylprolylchloromethylketone, andtosyllysl chloromethylketone. An illustrative heterocyclic proteaseinhibitor is an isocoumarin, and an exemplary transition state analogueis difluoroketomethylene. Inhibitors comprising a CCSDM moiety with theformula “X-C1-C2-A-Y” include a C1 component selected from the groupconsisting of β-homoarginine, an arginine containing a reducedcarboxylate moiety, and β-homoornithine. An illustrative arginine thatcontains a reduced carboxylate moiety is Argψ[CH₂NH]. Illustrativelinkers include linkers selected from the group consisting of: (i)A-L-[ED]-[ED]-X(1-3) (SEQ ID NO:131), (ii) A-L-X(1-3)-[ED]-[ED] (SEQ IDNO:132), (iii) A-L-[ED]-[ED] (SEQ ID NO:122), (iv) X(2-5)-[ED]-[ED] (SEQID NO:134), (v) A-L-[ED]-[ED]-X(1-2)-C (SEQ ID NO:136), (vi)A-L-[ED]-[ED]-C (SEQ ID NO:124), (vii) X(1-4)-[ED]-[ED]-C (SEQ IDNO:138), (viii) A-L-X(1-2)-[ED]-[ED]-C (SEQ ID NO:139), (ix) X(4-7) (SEQID NO:133), (x) X(5-7) (SEQ ID NO:135), and (xi) X(3-6)-C (SEQ IDNO:137),

[0028] where amino acid residues in square brackets indicate acceptableamino acids, numbers in parentheses indicate the number of amino acidresidues, and “X” represents any of glycine, serine, or threonine.

[0029] The present invention also contemplates complement C1sinhibitors, wherein the inhibitors consist of:

[0030] (a) a C1s catalytic site-directed moiety (CCSDM), which isselected from the group consisting of: (i) GCDGFKCRLG CTYGFKTDKKGCEAECTCNT (SEQ ID NO:53)

[0031]  and (ii) CRLGC (amino acid residues 64 to 68 of SEQ ID NO:1);

[0032] (b) a linker moiety characterized by a backbone chain having acalculated length of between 14 Å and 20 Å; and

[0033] (c) a C1s exosite binding moiety (CEBM), which is a polypeptidecharacterized by the formula:“A-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”where amino acid residues in square brackets indicate acceptable aminoacids, numbers in parentheses indicate the number of amino acidresidues, “X₁” represents Phe-(p-CH2)SO₃H, “X₂” represents sulfatedtyrosine, “X₃” represents 2-sulfotyrosine (SEQ ID NO:128);

[0034] wherein the C1s catalytic site-directed moiety is bound to thelinker moiety, the linker moiety is bound to the C1s exosite bindingmoiety.

[0035] Inhibitors comprising multiple functional moieties, as describedabove, can be characterized by the formula: “CCSDM-Linker-CEBM.” Thepresent invention also provides compositions that comprise a carrier,and a peptide, or a polypeptide, described herein, as well as methodsfor inhibiting complement C1s inhibitor, comprising the administrationof such compositions. These compositions can be administered to amammalian subject, such as a farm animal, a domestic animal, or a humanpatient.

[0036] These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

[0037] 2. Definitions

[0038] In the description that follows, a number of terms are usedextensively. The following definitions are provided to facilitateunderstanding of the invention.

[0039] As used herein, “nucleic acid” or “nucleic acid molecule” refersto polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), oligonucleotides, fragments generated by the polymerasechain reaction (PCR), and fragments generated by any of ligation,scission, endonuclease action, and exonuclease action. Nucleic acidmolecules can be composed of monomers that are naturally-occurringnucleotides (such as DNA and RNA), or analogs of naturally-occurringnucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Modified nucleotides can havealterations in sugar moieties and/or in pyrimidine or purine basemoieties. Sugar modifications include, for example, replacement of oneor more hydroxyl groups with halogens, alkyl groups, amines, and azidogroups, or sugars can be functionalized as ethers or esters. Moreover,the entire sugar moiety can be replaced with sterically andelectronically similar structures, such as aza-sugars and carbocyclicsugar analogs. Examples of modifications in a base moiety includealkylated purines and pyrimidines, acylated purines or pyrimidines, orother well-known heterocyclic substitutes. Nucleic acid monomers can belinked by phosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyarnide backbone. Nucleic acids can be either single stranded ordouble stranded.

[0040] The term “complement of a nucleic acid molecule” refers to anucleic acid molecule having a complementary nucleotide sequence andreverse orientation as compared to a reference nucleotide sequence.

[0041] The term “structural gene” refers to a nucleic acid molecule thatis transcribed into messenger RNA (mRNA), which is then translated intoa sequence of amino acids characteristic of a specific polypeptide.

[0042] An “isolated nucleic acid molecule” is a nucleic acid moleculethat is not integrated in the genomic DNA of an organism. For example, aDNA molecule that encodes a growth factor that has been separated fromthe genomic DNA of a cell is an isolated DNA molecule. Another exampleof an isolated nucleic acid molecule is a chemically-synthesized nucleicacid molecule that is not integrated in the genome of an organism. Anucleic acid molecule that has been isolated from a particular speciesis smaller than the complete DNA molecule of a chromosome from thatspecies.

[0043] A “nucleic acid molecule construct” is a nucleic acid molecule,either single- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature. “Linear DNA” denotesnon-circular DNA molecules having free 5′ and 3′ ends. Linear DNA can beprepared from closed circular DNA molecules, such as plasmids, byenzymatic digestion or physical disruption. “Complementary DNA (cDNA)”is a single-stranded DNA molecule that is formed from an mRNA templateby the enzyme reverse transcriptase. Typically, a primer complementaryto portions of mRNA is employed for the initiation of reversetranscription. Those skilled in the art also use the term “cDNA” torefer to a double-stranded DNA molecule consisting of such asingle-stranded DNA molecule and its complementary DNA strand. The term“cDNA” also refers to a clone of a cDNA molecule synthesized from an RNAtemplate.

[0044] A “promoter” is a nucleotide sequence that directs thetranscription of a structural gene. Typically, a promoter is located inthe 5′ non-coding region of a gene, proximal to the transcriptionalstart site of a structural gene. Sequence elements within promoters thatfunction in the initiation of transcription are often characterized byconsensus nucleotide sequences. These promoter elements include RNApolymerase binding sites, TATA sequences, CAAT sequences,differentiation-specific elements (DSEs; McGehee et al., Mol.Endocrinol. 7:551 (1993)), cyclic AMP response elements (CREs), serumresponse elements (SREs; Treisman, Seminars in Cancer Biol. 1:47(1990)), glucocorticoid response elements (GREs), and binding sites forother transcription factors, such as CRE/ATF (O'Reilly et al., J. Biol.Chem. 267:19938 (1992)), AP2 (Ye et al., J. Biol. Chem. 269:25728(1994)), SP1, cAMP response element binding protein (CREB; Loeken, GeneExpr. 3:253 (1993)) and octamer factors (see, in general, Watson et al.,eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/CummingsPublishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J.303:1 (1994)). If a promoter is an inducible promoter, then the rate oftranscription increases in response to an inducing agent. In contrast,the rate of transcription is not regulated by an inducing agent if thepromoter is a constitutive promoter. Repressible promoters are alsoknown.

[0045] A “core promoter” contains essential nucleotide sequences forpromoter function, including the TATA box and start of transcription. Bythis definition, a core promoter may or may not have detectable activityin the absence of specific sequences that may enhance the activity orconfer tissue specific activity.

[0046] A “regulatory element” is a nucleotide sequence that modulatesthe activity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

[0047] An “enhancer” is a type of regulatory element that can increasethe efficiency of transcription, regardless of the distance ororientation of the enhancer relative to the start site of transcription.“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

[0048] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides.” A “protein” is a macromolecule comprising one or morepolypeptide chains. A protein may also comprise non-peptidic components,such as carbohydrate groups. Carbohydrates and other non-peptidicsubstituents may be added to a protein by the cell in which the proteinis produced, and will vary with the type of cell. Proteins are definedherein in terms of their amino acid backbone structures; substituentssuch as carbohydrate groups are generally not specified, but may bepresent nonetheless.

[0049] A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

[0050] An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

[0051] A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, which has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

[0052] An “expression vector” is a nucleic acid molecule encoding a genethat is expressed in a host cell. Typically, an expression vectorcomprises a transcription promoter, a gene, and a transcriptionterminator. Gene expression is usually placed under the control of apromoter, and such a gene is said to be “operably linked to” thepromoter. Similarly, a regulatory element and a core promoter areoperably linked if the regulatory element modulates the activity of thecore promoter.

[0053] A “recombinant host” is a cell that contains a heterologousnucleic acid molecule, such as a cloning vector or expression vector. Inthe present context, an example of a recombinant host is a cell thatproduces a complement C1s inhibitory peptide or polypeptide from anexpression vector.

[0054] “Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

[0055] A “fusion protein” is a hybrid protein expressed by a nucleicacid molecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a complement C1sinhibitor fused with a polypeptide that binds an affinity matrix. Such afusion protein provides a means to isolate large quantities of acomplement C1s inhibitor using affinity chromatography.

[0056] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

[0057] In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

[0058] The term “secretory signal sequence” denotes a nucleotidesequence that encodes a peptide (a “secretory peptide”) that, as acomponent of a larger polypeptide, directs the larger polypeptidethrough a secretory pathway of a cell in which it is synthesized. Thelarger polypeptide is commonly cleaved to remove the secretory peptideduring transit through the secretory pathway.

[0059] An “isolated polypeptide” is a polypeptide that is essentiallyfree from contaminating cellular components, such as carbohydrate,lipid, or other proteinaceous impurities associated with the polypeptidein nature. Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

[0060] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0061] The term “expression” refers to the biosynthesis of a geneproduct. For example, in the case of a structural gene, expressioninvolves transcription of the structural gene into mRNA and thetranslation of mRNA into one or more polypeptides.

[0062] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. If subsequentdissociation of the complement/anti-complement pair is desirable, thenthe complement/anti-complement pair preferably is characterized by abinding affinity of less than 10⁹ M⁻¹.

[0063] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). Nucleic acid molecules encoding affinity tagsare available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

[0064] Due to the imprecision of standard analytical methods, molecularweights and lengths of polymers are understood to be approximate values.When such a value is expressed as “about” X or “approximately” X, thestated value of X will be understood to be accurate to ±10%.

[0065] 3. Synthetic Complement C1s Inhibitory Peptides and Polypeptides

[0066] A. C1s Exosite Binding Moiety

[0067] One series of peptides, designed to bind the exosite region ofC1s, are derived from the C-terminus of BD001, or the C-terminus of C4.These peptides may include tyrosine residues that lack sulfation, or oneor more tyrosine residues that are sulfated. If tyrosine sulfation isdesired, then peptides can contain either sulfated tyrosine or an analogof sulfated tyrosine. An example of a tyrosine sulfate analog isPhe-(p-CH₂)SO₃H. Methods for synthesizing this analog are known to thoseof skill in the art (see, for example, Gonzalez-Muniz et al., Int. J.Peptide Protein Res. 37:331 (1991)).

[0068] The following formula describes one suitable class of C1s exositebinding moieties: “[AP]-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),” where amino acid residues insquare brackets indicate acceptable amino acids, numbers in parenthesesindicate the number of amino acid residues, “X₁” represents sulfatedphenylalanine (Phe-(p-CH₂)SO₃H), “X₂” represents sulfated tyrosine(Tyr(OSO₃H)), and “X₃” represents 2-sulfotyrosine (SEQ ID NO:126).Examples of C1s exosite binding polypeptides are provided in Table 1 andTable 2. Such polypeptides can include tyrosine residues that arenon-sulfated or sulfated. Those of skill in the art using the aboveformula can devise additional polypeptides. The activity of thesepolypeptides can be assessed by measuring the ability to inhibit aclassical complement hemolysis assay, such as the assay presented inExample 1.

[0069] The compounds referred to as sulfated phenylalanine, sulfatedtyrosine, and 2-sulfotyrosine have the structural formulae:

TABLE 1 Amino Acid Sequence¹ SEQ ID NO P-N-E-E-Y-E-Y-D-Y-E  2P-N-E-E-X₁-E-Y-D-Y-E  3 P-N-E-E-Y-E-X₁-D-Y-E  4 P-N-E-E-Y-E-Y-D-X₁-E  5P-N-E-E-X₁-E-X₁-D-Y-E  6 P-N-E-E-X₁-E-Y-D-X₁-E  7 P-N-E-E-Y-E-X₁-D-X₁-E 8 P-N-E-E-X₁-E-X₁-D-X₁-E  9 P-N-E-E-X₂-E-Y-D-Y-E 10P-N-E-E-Y-E-X₂-D-Y-E 11 P-N-E-E-Y-E-Y-D-X₂-E 12 P-N-E-E-X₂-E-X₂-D-Y-E 13P-N-E-E-X₂-E-Y-D-X₂-E 14 P-N-E-E-Y-E-X₂-D-X₂-E 15 P-N-E-E-X₂-E-X₂-D-X₂-E16 P-N-E-E-X₁-E-X₂-D-Y-E 17 P-N-E-E-X₂-E-X₁-D-Y-E 18P-N-E-E-X₁-E-Y-D-X₂-E 19 P-N-E-E-X₂-E-Y-D-X₁-E 20 P-N-E-E-Y-E-X₁-D-X₂-E21 P-N-E-E-Y-E-X₂-D-X₁-E 22 P-N-E-E-X₁-E-X₁-D-X₂-E 23P-N-E-E-X₂-E-X₁-D-X₁-E 24 P-N-E-E-X₁-E-X₂-D-X₂-E 25P-N-E-E-X₁-E-X₂-D-X₁-E 26 P-N-E-E-X₂-E-X₂-D-X₁-E 27

[0070] TABLE 2 Amino Acid Sequence¹ SEQ ID NO A-N-E-D-X₁-E-D-Y-E-Y-D 28A-N-E-D-Y-E-D-X₁-E-Y-D 29 A-N-E-D-Y-E-D-Y-E-X₁-D 30A-N-E-D-X-E-D-X₁-E-Y-D 31 A-N-E-D-X₁-E-D-Y-E-X₁-D 32A-N-E-D-Y-E-D-X₁-E-X₁-D 33 A-N-E-D-X₁-E-D-X₁-E-X₁-D 34A-N-E-D-X₂-E-D-Y-E-Y-D 35 A-N-E-D-Y-E-D-X₂-E-Y-D 36A-N-E-D-Y-E-D-Y-E-X₂-D 37 A-N-E-D-X₂-E-D-X₂-E-Y-D 38A-N-E-D-X₂-E-D-Y-E-X₂-D 39 A-N-E-D-Y-E-D-X₂-E-X₂-D 40A-N-E-D-X₂-E-D-X₂-E-X₂-D 41 A-N-E-D-X₁-E-D-X₂-E-Y-D 42A-N-E-D-X₂-E-D-X₁-E-Y-D 43 A-N-E-D-X₁-E-D-Y-E-X₂-D 44A-N-E-D-X₂-E-D-Y-E-X₁-D 45 A-N-E-D-Y-E-D-X₁-E-X₂-D 46A-N-E-D-Y-E-D-X₂-E-X₁-D 47 A-N-E-D-X₁-E-D-X₁-E-X₂-D 48A-N-E-D-X₂-E-D-X₁-E-X₁-D 49 A-N-E-D-X₁-E-D-X₂-E-X₂-D 50A-N-E-D-X₁-E-D-X₂-E-X₁-D 51 A-N-E-D-X₂-E-D-X₂-E-X₁-D 52

[0071] B. C1s Catalytic Site-directed Moiety

[0072] Studies were performed to localize the active site of BD001. Inone group of experiments, BD001 was incubated with C1s to cleave BD001,and then cleaved BD001 was fractionated using SDS-polyacrylamide gelelectrophoresis. Fractionated protein fragments were thenelectro-blotted onto nitrocellulose, and sequenced using standard Edmandegradation. The results revealed the presence of fragments consistingof an amino acid sequence that included the N-terminus of BD001 to aminoacid residue 66. Åccordingly, one type of C1s catalytic site-directedmoiety is a peptide or polypeptide comprising amino acid residues 64 to68 of SEQ ID NO:1. Ån illustrative polypeptide comprises the followingamino acid sequence: GCDGFKCRLG CTYGFKTDKK GCEAFCTCNT (SEQ ID NO:53).

[0073] Additional amino acid sequences are presented in Table 3. Certainof these C1s catalytic site-directed moieties are variations of SEQ IDNO:53 in which cysteine residues have been replaced by serine residues.In particular embodiments, these serine-substituted polypeptides areoxidized to induce the formation of disulfide bonds. TABLE 3 Amino AcidSequence SEQ ID NO GCDGFKCRLGCTYGFKTDKKGCEAFCTCNT 53GCDGFKSRLGSTYGFKTDKKGCEAFSTSNT 54 GSDGFKCRLGSTYGFKTDKKGSEAFCTSNT 55GSDGFKSRLGCTYGFKTDKKGSEAFSTCNT 56 GCDGFKSRLGCTYGFKTDKKGCEAFCTCNT 57GCDGFKCRLGSTYGFKTDKKGCEAFCTCNT 58 GCDGFKCRLGCTYGFKTDKKGCEAFSTCNT 59GCDGFKCRLGCTYGFKTDKKGCEAFCTSNT 60 GCDGFKSRLGSTYGFKTDKKGCEAFCTCNT 61GCDGFKSRLGCTYGFKTDKKGCEAFSTCNT 62 GCDGFKSRLGCTYGFKTDKKGCEAECTSNT 63GCDGFKCRLGSTYGFKTDKKGCEAFSTCNT 64 GCDGFKCRLGSTYGFKTDKKGCEAECTSNT 65GCDGFKCRLGCTYGFKTDKKGCEAFSTSNT 66 GCDGFKSRLGSTYGFKTDKKGCEAFSTCNT 67GCDGFKSRLGSTYGFKTDKKGCEAFCTSNT 68 GCDGFKCRLGSTYGFKTDKKGCEAFSTSNT 69GCDGFKSRLGCTYGFKTDKKGCEAFSTSNT 70 GSDGFKCRLGCTYGFKTDKKGCEAFCTCNT 71GCDGFKCRLGSTYGFKTDKKGCEAFCTCNT 72 GCDGFKCRLGCTYGFKTDKKGSEAFCTCNT 73GCDGFKCRLGCTYGFKTDKKGCEAFCTSNT 74 GSDGFKCRLGSTYGFKTDKKGCEAECTCNT 75GSDGFKCRLGCTYGFKTDKKGSEAFCTCNT 76 GSDGFKCRLGCTYGFKTDKKGCEAFCTSNT 77GCDGFKCRLGSTYGFKTDKKGSEAFCTCNT 78 GCDGFKCRLGSTYGFKTDKKGCEAFCTSNT 79GCDGFKCRLGCTYGFKTDKKGSEAFCTSNT 80 GSDGFKCRLGSTYGFKTDKKGSEAECTCNT 81GSDGFKCRLGCTYGFKTDKKGSEAFCTSNT 82 GCDGFKCRLGSTYGFKTDKKGSEAFCTSNT 83GSDGFKCRLGSTYGFKTDKKGCEAECTSNT 84 GSDGFKCRLGCTYGFKTDKKGCEAFCTCNT 85GCDGFKSRLGCTYGFKTDKKGCEAFCTCNT 86 GCDGFKCRLGCTYGFKTDKKGSEAECTCNT 87GCDGFKCRLGCTYGFKTDKKGCEAFSTCNT 88 GSDGFKSRLGCTYGFKTDKKGCEAECTCNT 89GSDGFKCRLGCTYGFKTDKKGSEAFCTCNT 90 GSDGFKCRLGCTYGFKTDKKGCEAFSTCNT 91GCDGFKSRLGCTYGFKTDKKGSEAFCTCNT 92 GCDGFKSRLGCTYGFKTDKKGCEAESTCNT 93GCDGFKCRLGCTYGFKTDKKGSEAFSTCNT 94 GSDGFKSRLGCTYGFKTDKKGSEAECTCNT 95GSDGFKSRLGCTYGFKTDKIKGCEAESTCNT 96 GCDGFKSRLGCTYGFKTDKKGSEAFSTCNT 97GSDGFKCRLGCTYGFKTDKKGSEAFSTCNT 98+TZ,1/32

[0074] A C1s catalytic site-directed moiety can also consist of theamino acid sequence CRLGC (amino acid residues 64 to 68 of SEQ ID NO:1).In addition, a C1s catalytic site-directed moiety can comprise a peptideor polypeptide shown in Table 4. Those of skill in the art can devisefurther modifications of the sequences disclosed herein. TABLE 4 AminoAcid Sequence SEQ ID NO CRLGCT 99 CRLGCTY 100 CRLGCTYG 101 CRLGCTYGF 102CRLGCTYGFK 103 CRLGCTYGFKT 104 CRLGCTYGFKTD 105 CRLGCTYGFKTDK 106CRLGCTYGFKTDKK 107 CRLGCTYGFKTDKKG 108 CRLGCTYGFKTDKKGC 109CRLGCTYGFKTDKKGCE 110 CRLGCTYGFKTDKKGCEA 111 CRLGCTYGFKTDKKGCEAF 112CRLGCTYGFKTDKKGCEAFC 113 CRLGCTYGFKTDKKGCEAECT 114CRLGCTYGFKTDKKGCEAECTC 115 CRLGCTYGFKTDKKGCEAECTCN 116

[0075] C. Bivalent Polypeptide Inhibitors

[0076] Bivalent polypeptide inhibitors comprise a C1s catalyticsite-directed moiety, a linker, and a C1s exosite binding moiety.Various C1s catalytic site-directed moieties, described above, can beused for bivalent inhibitors. Additional useful catalytic site-directedmoieties include molecules that are cleaved by C1s. For example,Pefachrome C1E (Pentapharm; Basel, Switzerland) is a para-nitroaniline(pNA) containing substrate, which is cleaved by C1s to release free pNA.Pefachrome C1E has the following sequence, in which “Cbo” representsbenzyloxycarbonyl: CH₃-Lys(Cbo)-Gly-Arg-pNA-AcOH. A derivative ofPefachrome C1E that would be suitable as a catalytic site-directedmoiety is: CH₃-Lys(Cbo)-Gly-Arg. As another example, S-2314(Chromogenix; Milano, Italy) is a colorimetric substrate, which iscleaved by C1s, and which has the following sequence:H-D-Val-Ser-Arg-pNA.HCl. A derivative of this molecule, which would besuitable as a catalytic site-directed moiety is: H-D-Val-Ser-Arg.

[0077] Those of skill in the art can devise additional catalyticsite-directed moieties from known C1s substrates. As an illustration,the following sequence appears to be a recognition site in human C4 thatconfers specificity for C1s: LQRALE (SEQ ID NO:117).

[0078] The cleavage site is located between the Arg and Ala, and thedownstream residues appear to be important for recognition by C1s (see,for example, Ogata et al., Proc. Nat'l Acad. Sci. USA. 86:5575 (1989),and Ogata and Low, J. of Immunol. 155:2642 (1995)). Based upon studieswith closely related complement proteins C3, C4, C5, and sex-limitedprotein, researchers have suggested that the minimally required activesite sequence is: Leu-Xaa-Arg, where Xaa is Ala, Gln, or Gly.Accordingly, such sequences can provide suitable C1s catalytic-sitedirected moieties.

[0079] Researchers have shown that the cleavage site of C4 can inhibitthe classical complement pathway when linked to the C-terminal portionof antithrombin III (Glover et al., Molec. Immunol. 25:1261 (1988);Schasteen et al., Molec. Immunol. 25:1269 (1988)). The polypeptide,which has the sequence LQRALEILPN RVTIKANRPF LVFI (SEQ ID NO:118),

[0080] is another suitable C1s catalytic site-directed moiety.

[0081] Catalytic site-directed moieties can be designed to bindirreversibly to C1s protease. Examples of such irreversible active siteinhibitors include general serine protease inhibitors (e.g.,phenylmethylsulfonylfluoride, diisopropylflouorophosphate,tosylprolylchloromethylketone, tosyllysl chloromethylketone, etc.),heterocyclic protease inhibitors, such as isocoumarins, and transitionstate analogues, such as difluoroketomethylene.

[0082] Another type of catalytic site-directed moiety can consist ofnon-cleavable, reversible active site inhibitors. One example of auseful non-cleavable reversible active site inhibitor is benzamidine. Asanother example, inhibitors can be characterized by the formula:X-C1-C2-A-Y, where C1 is a derivative of Arg, Lys, or Orn, characterizedby a reduced carboxylate moiety or a carboxylate moiety that isdisplaced from the α-carbon by a chemical structure characterized by abackbone chain of from 1-10 atoms; C2 is a non-cleavable bond; X ishydrogen or a continuation of the peptide backbone; A is a backbonechain; and Y is a bond. Examples of Cl components includeβ-homoarginine; arginine containing a reduced carboxylate moiety, suchas Argψ[CH₂NH]; β-homolysine; and β-homoomithine. Methods forsynthesizing such analogues are known to those of skill in the art. Forexample, Steinmetzer et al., J. Med. Chem. 42:3109 (1999), describemethods for incorporating various arginyl ketomethylene isosteres(Argψ[CO-CH₂-X]P₁′) into polypeptides as P₁-P₁′ segments to eliminatethe scissile bond, where Pl′ can be a natural or an unnatural aminoacid.

[0083] Illustrative C1s exosite binding moieties include the moleculesdescribed above, as well as the polypeptide NEDYEDYEYD (SEQ ID NO:119).

[0084] Computer modeling studies revealed that suitable linkers ofbivalent polypeptide inhibitors have a backbone chain with a calculatedlength of about 14 Å to about 20 Å, about 15 Å to about 19 Å, or about16 Å to about 18 Å (e.g., 14 Å to 20 A, 15 Å to 19 Å, or 16 Å to 18 Å).The term “backbone chain” refers to the portion of a chemical structurethat defines the smallest number of consecutive bonds that can be tracedfrom one end of the structure to the other end. A backbone chain cancomprise atoms capable of forming bonds with at least two other atoms.The term “calculated length” refers to a measurement derived by summingup the bond lengths between the atoms, which comprise the backbonechain. Linkers are also contemplated that include certain proteindomains, such as a linker comprising the amino acid sequence “KETACVNIWCTDPYKCNPES GRC” (SEQ ID NO:120).

[0085] Suitable linkers include peptides comprised of about two to aboutnine amino acids, about four to about seven amino acids, about five toseven amino acids, or six to seven amino acids. Illustrativecombinations of C1s catalytic site-directed moieties and linkers areprovided in Table 5. TABLE 5 +HZ,1/41 C1s catalytic site-directedmoiety¹ Linker¹ L-Q-R A-L-[ED]-[ED]-X(1-3) (SEQ ID NO:131) BenzamidineA-L-X(1-3)-[ED]-[ED] (SEQ ID NO:132) A-L-[ED]-[ED](SEQ ID NO:122) X(4-7)(SEQ ID NO:133) X(2-5)-[ED]-[ED](SEQ ID NO:134)C-D-G-F-K-C-R-L-G-C-T-Y-G-F-K- K-E-T-A-C-V-N-I-W-C-T-D-P-YT-D-K-K-G-C-E-A-F-C-T-C-N-T K-C-N-P-E-S-G-R-C-E-D (SEQ ID NO:121) (SEQID NO:123) X(5-7) (SEQ ID NO:135) A-L-[ED]-[ED]-X(1-3) (SEQ ID NO:131)A-L-X(1-3)-[ED]-[ED] (SEQ ID NO:132) X-C-X(8-12)-L-Q-RA-L-[ED]-[ED]-X(1-2)-C (SEQ ID NO:140) (SEQ ID NO:136) A-L-[ED]- [ED]-C(SEQ ID NO:124) X(3-6)-C (SEQ ID NO:137) X(1-4)-[ED]-[ED]-C (SEQ IDNO:138) A-L-X(1-2)-[ED]-[ED]-C (SEQ ID NO:139)+TZ,1/41

[0086] Bivalent polypeptide inhibitors can be produced synthetically orrecombinantly, as described below. Alternatively, bivalent polypeptideinhibitors can be assembled by conjugating synthetically- orrecombinantly-produced C1s catalytic site-directed and C1s exositebinding moieties with linkers. Well-known methods of conjugatingpolypeptides are described by Lappi et al., Biochem. Biophys. Res.Commun. 160:917 (1989), Wong, Chemistry of Protein Conjugation andCross-Linking (CRC Press 1991), Soria et al., Targeted Diagn. Ther.7:193 (1992), Buechler et al., Eur. J. Biochem. 234:706 (1995),Behar-Cohen et al., Invest. Ophthalmol. Vis. Sci. 36:2434 (1995), Lappiand Baird, U.S. Pat. No. 5,191,067, Calabresi et al., U.S. Pat. No.5,478,804, and Lappi and Baird, U.S. Pat. No. 5,576,288. Ådditionalapproaches to conjugating polypeptides are known to those of skill inthe art.

[0087] In certain embodiments, either the carboxy-terminus or theamino-terminus, or both, are chemically modified. For example, terminalamino groups can be acetylated, whereas carboxyl groups can be amidated.Amino-terminal modifications such as acylation (e.g., acetylation) oralkylation (e.g., methylation) and carboxy-terminal modifications suchas amidation, as well as other terminal modifications, includingcyclization, may be incorporated into peptides and polypeptidesdescribed herein.

[0088] The present invention also includes complement inhibitors that donot comprise only naturally occurring amino acids. In general, such“peptidomimetics” are structurally similar to a model inhibitory peptideor polypeptide, but have one or more peptide linkages optionallyreplaced by a linkage such as: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CHCH— (cisand trans), —COCH2—, —CH(OH)CH₂—, —CH₂SO—, and the like. Methods forpreparing such polypeptide analogs are known to those of skill in theart (see, for example, Kazmierski (Ed.), Peptidomimetics Protocols(Humana Press, Inc. 1998); Abel (Ed.), Advances in Amino Acid Mimeticsand Peptidomimetics (JAI Press, 1999)).

[0089] Examples of suitable non-naturally occurring amino acids includenorleucine, alloisoleucine, homoarginine, thiaproline, dehydroproline,homoserine, cyclohexylglycine-amino-n-butyric acid, cyclohexylalanine,aminophenylbutyric acid, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, 4-fluorophenylalanine,phenylalanines substituted at the ortho, meta, or para position of thephenyl moiety with one or two of the following: a (C₁-C₄) alkyl, a(C₁-C₄) alkoxy, halogen, or nitro groups or substituted with amethylenedioxy group; β-2- and 3-thienylalanine, β-2- and3-furanylalanine, β-2-, 3- and 4-pyridylalanine, β-(benzothienyl-2- and3yl)alanine, β-(1- and 2-naphthyl)alanine, O-alkylated derivatives ofserine, threonine, or tyrosine, S-alkylated cysteine, S-alkylatedhomocysteine, O-sulfate, O-phosphate, and O-carboxylate esters oftyrosine, 3- and 5-sulfonyl tyrosine, 3- and 5-carbonyl tyrosine, 3- and5-phosphonyl tyrosine, 4-methylsulfonyl tyrosine, 4-methylphosphonyltyrosine, 4-phenylacetic acid, 3,5-diiodotyrosine, 3- and5-nitrotyrosine, ε-alkyl lysine, delta-alkyl ornithine, and the like, aswell as D-isomers of the naturally occurring amino acids.

[0090] Several methods are known in the art for incorporatingnon-naturally occurring amino acid residues into proteins. For example,an in vitro system can be employed wherein nonsense mutations aresuppressed using chemically aminoacylated suppressor tRNAs. Methods forsynthesizing amino acids and aminoacylating tRNA are known in the art.Transcription and translation of plasmids containing nonsense mutationsis typically carried out in a cell-free system comprising an E. coli S30extract and commercially available enzymes and other reagents. Proteinsare purified by chromatography. See, for example, Robertson et al., J.Am. Chem. Soc. 113:2722 (1991), Ellman et al., Methods Enzymol. 202:301(1991), Chung et al., Science 259:806 (1993), and Chung et aL, Proc.Nat'l Acad. Sci. USA 90:10145 (1993).

[0091] Those of skill in the art devise additional variations of C1sexosite binding moieties, linkers, and C1s catalytic site-directedmoieties described herein. For example, a systematic substitution of oneor more amino acids with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) can be used to generate more stable peptides. Inaddition, constrained peptides (including cyclized peptides) can begenerated by methods known in the art (see, for example, Rizo andGierasch, Ann. Rev. Biochem. 61: 387 (1992)).

[0092] The biological activity of C1s exosite binding moieties, C1scatalytic site-directed moieties, and bivalent inhibitor polypeptidescan be tested in a variety of assays. Example 1 illustrates severalapproaches. In addition, a simple standard assay for C1-esteraseinhibitor activity can be based upon the ability of a test substance toblock the production of a chromogenic proteolytic product following theaddition of purified C1s to plasma, as described by Wiman and Nilsson,Clin. Chem. Acta 128:359 (1983). Another standard approach requires thekinetic measurement of a putative C1s inhibitor as it inhibits thehydrolysis of N-acetyl-L-tyrosine-ethyl ester by C1 esterase (Schena etal., J. Clin. Chem. Clin. Biochem. 18:17 (1980)). Additional assays canbe devised by those of skill in the art.

[0093] 4. Chemical Synthesis and Semi-synthesis of Complement C1sInhibitory Peptides and Polypeptides

[0094] Inhibitory peptides and polypeptides of the present invention canbe synthesized using standard techniques, including solid phasesynthesis, partial solid phase methods, fragment condensation, orclassical solution synthesis. The polypeptides can be prepared by solidphase peptide synthesis, for example as described by Merrifield, J. Am.Chem. Soc. 85:2149 (1963). The synthesis is carried out with amino acidsthat are protected at the α-amino terminus. Trifunctional amino acidswith labile side-chains are also protected with suitable groups toprevent undesired chemical reactions from occurring during the assemblyof the polypeptides. The α-amino protecting group is selectively removedto allow subsequent reaction to take place at the amino-terminus. Theconditions for the removal of the α-amino protecting group do not removethe side-chain protecting groups.

[0095] The α-amino protecting groups are those known to be useful in theart of stepwise polypeptide synthesis. Included are acyl type protectinggroups (e.g., formyl, trifluoroacetyl, acetyl), aryl type protectinggroups (e.g., biotinyl), aromatic urethane type protecting groups [e.g.,benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and9-fluorenylmethyloxy-carbonyl (Fmoc)], aliphatic urethane protectinggroups [e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,cyclohexloxycarbonyl] and alkyl type protecting groups (e.g., benzyl,triphenylmethyl). The preferred protecting groups are tBoc and Fmoc,thus the peptides are said to be synthesized by tboc and Fmoc chemistry,respectively.

[0096] The side-chain protecting groups selected must remain intactduring coupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

[0097] In tBoc chemistry, the preferred side-chain protecting groups aretosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (andacetamidomethyl) for cysteine, benzyl for glutamic acid, serine andthreonine, benzyloxymethyl (and dinitrophenyl) for histidine,2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chainprotecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,trityl for asparagine, cysteine, glutarnine and histidine, tert-butylfor aspartic acid, glutamic acid, serine, threonine and tyrosine, tbocfor lysine and tryptophan.

[0098] For the synthesis of phosphopeptides, either direct orpost-assembly incorporation of the phosphate group is used. In thedirect incorporation strategy, the phosphate group on serine, threonineor tyrosine may be protected by methyl, benzyl, or tert-butyl in Fmocchemistry or by methyl, benzyl or phenyl in tBoc chemistry. Directincorporation of phosphotyrosine without phosphate protection can alsobe used in Fmoc chemistry. In the post-assembly incorporation strategy,the unprotected hydroxyl groups of serine, threonine or tyrosine arederivatized on solid phase with di-tert-butyl-, dibenzyl- ordimethyl-N,N′-diisopropylphosphorarnidite and then oxidized bytert-butylhydroperoxide.

[0099] Solid phase synthesis is usually carried out from thecarboxyl-terminus by coupling the alpha-amino protected (side-chainprotected) amino acid to a suitable solid support. An ester linkage isformed when the attachment is made to a chloromethyl, chlortrityl orhydroxymethyl resin, and the resulting polypeptide will have a freecarboxyl group at the C-terminus. Alternatively, when an amide resinsuch as benzhydrylamine or p-methylbenzhydrylamine resin (for tBocchemistry) and Rink amide or PAL resin (for Fmoc chemistry) are used, anamide bond is formed and the resulting polypeptide will have acarboxamide group at the C-terminus. These resins, whether polystyrene-or polyamide-based or polyethyleneglycol-grafted, with or without ahandle or linker, with or without the first amino acid attached, arecommercially available, and their preparations have been described byStewart et al., “Solid Phase Peptide Synthesis” (2nd Edition), (PierceChemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986),Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach(IRL Press 1989), and by Lloyd-Williams et al., Chemical Approaches tothe Synthesis of Peptides and Proteins (CRC Press, Inc. 1997).

[0100] The C-terminal amino acid, protected at the side chain ifnecessary, and at the alpha-amino group, is attached to a hydroxylmethylresin using various activating agents including dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole(CDI). It can be attached to chloromethyl or chlorotrityl resin directlyin its cesium tetramethylammonium salt form or in the presence oftriethylamine (TEA) or diisopropylethylamine (DIEA). First amino acidattachment to an amide resin is the same as amide bond formation duringcoupling reactions.

[0101] Following the attachment to the resin support, the α-aminoprotecting group is removed using various reagents depending on theprotecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal canbe monitored at 300-320 nm or by a conductivity cell. After removal ofthe alpha-amino protecting group, the remaining protected amino acidsare coupled stepwise in the required order to obtain the desiredsequence.

[0102] Various activating agents can be used for the coupling reactionsincluding DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate(CIP), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog(HBPyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) orits pyrrolidine analog (HAPyU). The most common catalytic additives usedin coupling reactions include 4-dimethylaminopyridine (DMAP),3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).Each protected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH2Cl2 or mixtures thereof. The extent of completion of thecoupling reaction can be monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., Anal. Biochem. 34:595 (1970). Incases where incomplete coupling is found, the coupling reaction isextended and repeated and may have chaotropic salts added. The couplingreactions can be performed automatically with commercially availableinstruments such as ABI model 430A, 431A and 433A peptide synthesizers.

[0103] After the entire assembly of the desired peptide, thepeptide-resin is cleaved with a reagent with proper scavengers. The Fmocpeptides are usually cleaved and deprotected by TFA with scavengers(e.g., water, ethanedithiol, phenol and thioanisole). The tBoc peptidesare usually cleaved and deprotected with liquid HF for 1-2 hours at −5to 0° C., which cleaves the polypeptide from the resin and removes mostof the side-chain protecting groups. Scavengers such as anisole,dimethylsulfide and p-thiocresol are usually used with the liquid HF toprevent cations formed during the cleavage from alkylating and acylatingthe amino acid residues present in the polypeptide. The formyl group oftryptophan and the dinitrophenyl group of histidine need to be removed,respectively by piperidine and thiophenyl in DMF prior to the HFcleavage. The acetamidomethyl group of cysteine can be removed bymercury(II)acetate and alternatively by iodine,thallium(III)trifluoroacetate or silver tetrafluoroborate whichsimultaneously oxidize cysteine to cystine. Other strong acids used fortBoc peptide cleavage and deprotection include trifluoromethanesulfonicacid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).

[0104] The “native chemical ligation” approach to producing polypeptidesis one variation of total chemical synthesis strategy (see, for example,Dawson et al., Science 266:776 (1994), Hackeng et al., Proc. Nat'l Acad.Sci. USA 94:7845 (1997), and Dawson, Methods Enzymol. 287: 34 (1997)).According to this method, an N-terminal cysteine-containing peptide ischemically ligated to a peptide having a C-terminal thioester group toform a normal peptide bond at the ligation site.

[0105] The “expressed protein ligation” method is a semi-synthesisvariation of the ligation approach (see, for example, Muir et al, Proc.Nat'l Acad. Sci. USA 95:6705 (1998); Severinov and Muir, J. Biol. Chem.273:16205 (1998)). Here, synthetic peptides and protein cleavagefragments are linked to form the desired protein product. This method isparticularly useful for the site-specific incorporation of unnaturalamino acids (e.g., amino acids comprising biophysical or biochemicalprobes) into proteins.

[0106] In an approach illustrated by Muir et al, Proc. Nat'l Acad. Sci.USA 95:6705 (1998), a gene or gene fragment is cloned into thePCYB2-IMPACT vector (New England Biolabs, Inc.; Beverly, Mass.) usingthe NdeI and SmaI restriction sites. As a result, the gene or genefragment is expressed in frame fused with a chitin binding domainsequence, and a Pro-Gly is appended to the native C terminus of theprotein of interest. The presence of a C-terminal glycine reduces thechance of side reactions, because the glycine residue accelerates nativechemical ligation. Affinity chromatography with a chitin resin is usedto purify the expressed fusion protein, and the chemical ligation stepis initiated by incubating the resin-bound protein with thiophenol andsynthetic peptide in buffer. This mixture produces the in situgeneration of a highly reactive phenyl ^(α)thioester derivative of theprotein that rapidly ligates with the synthetic peptide to produce thedesired semi-synthetic protein. For a review, see Kochendoerfer andKent, Curr. Opin. Chem. Biol. 3:665 (1999).

[0107] In an alternative approach, peptides and polypeptides can beproduced using combinatorial chemistry to synthesize a library ofanalogs for all positions of the desired peptide or polypeptide. See,for example, Gershengorn et al., international publication No. WO98/34948, Hruby et al., Curr. Opin. Chem. Biol. 1:114 (1997), andal-Obeidi et al., Mol. Biotechnol. 9:205 (1998).

[0108] 5. Recombinant Production of Complement C1s Inhibitor Peptidesand Polypeptides

[0109] The peptides and polypeptides of the present can also be producedin recombinant host cells following conventional techniques. Nucleicacid molecules that encode a C1s exosite binding moiety, a C1s catalyticsite-directed moiety, or a bivalent inhibitor polypeptide cansynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes, however, special strategies may be required, because thecoupling efficiency of each cycle during chemical DNA synthesis isseldom 100%. To overcome this problem, synthetic genes (double-stranded)are assembled in modular form from single-stranded fragments that arefrom 20 to 100 nucleotides in length. For reviews on polynucleotidesynthesis, see, for example, Glick and Pasternak, MolecularBiotechnology, Principles and Applications of Recombinant DNA (ASM Press1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), and Climie etal., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

[0110] To express a complement C1s inhibitor peptide or polypeptideencoding sequence, a nucleic acid molecule encoding the peptide orpolypeptide must be operably linked to regulatory sequences that controltranscriptional expression in an expression vector and then, introducedinto a host cell. In addition to transcriptional regulatory sequences,such as promoters and enhancers, expression vectors can includetranslational regulatory sequences and a marker gene, which is suitablefor selection of cells that carry the expression vector.

[0111] Expression vectors that are suitable for production of a foreignprotein in eukaryotic cells typically contain (1) prokaryotic DNAelements coding for a bacterial replication origin and an antibioticresistance marker to provide for the growth and selection of theexpression vector in a bacterial host; (2) eukaryotic DNA elements thatcontrol initiation of transcription, such as a promoter; and (3) DNAelements that control the processing of transcripts, such as atranscription termination/polyadenylation sequence. Expression vectorscan also include nucleotide sequences encoding a secretory sequence thatdirects the heterologous polypeptide into the secretory pathway of ahost cell.

[0112] Complement C1s inhibitor peptides and polypeptides of the presentinvention may be expressed in mammalian cells. Examples of suitablemammalian host cells include African green monkey kidney cells (Vero;ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573),baby hamster kidney cells (BHK-21, BHK-570; ÅTCC CRL 8544, ÅTCC CRL10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovarycells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin et al., Som. Cell. Molec.Genet. 12:555 1986]), rat pituitary cells (GHl; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548)SV40-transformed monkey kidney cells (COS-1; ÅTCC CRL 1650) and murineembryonic cells (NIH-3T3; ÅTCC CRL 1658).

[0113] For a mammalian host, the transcriptional and translationalregulatory signals may be derived from viral sources, such asadenovirus, bovine papilloma virus, simian virus, or the like, in whichthe regulatory signals are associated with a particular ene which has ahigh level of expression. Suitable transcriptional and translationalregulatory sequences also can be obtained from mammalian genes, such asactin, collagen, myosin, and metallothionein genes.

[0114] Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

[0115] Alternatively, a prokaryotic promoter, such as the bacteriophageT3 RNA polymerase promoter, can be used to control expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman etal., Nucl. Acids Res. 19:4485 (1991)).

[0116] An expression vector can be introduced into host cells using avariety of standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

[0117] For example, one suitable selectable marker is a gene thatprovides resistance to the antibiotic neomycin. In this case, selectionis carried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

[0118] Complement C1s inhibitor peptides and polypeptides can also beproduced by cultured mammalian cells using a viral delivery system.Exemplary viruses for this purpose include adenovirus, herpesvirus,vaccinia virus and adeno-associated virus (AAV). Adenovirus, adouble-stranded DNA virus, is currently the best studied gene transfervector for delivery of heterologous nucleic acid (for a review, seeBecker et al., Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel,Science & Medicine 4:44 (1997)). Advantages of the adenovirus systeminclude the accommodation of relatively large DNA inserts, the abilityto grow to high-titer, the ability to infect a broad range of mammaliancell types, and flexibility that allows use with a large number ofavailable vectors containing different promoters.

[0119] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

[0120] Nucleic acid molecules encoding complement C1s inhibitor peptidesand polypeptides may also be expressed in other higher eukaryotic cells,such as avian, fungal, insect, yeast, or plant cells. The baculovirussystem provides an efficient means to introduce cloned complement C1sinhibitor genes into insect cells. Suitable expression vectors are basedupon the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV), and contain well-known promoters such as Drosophila heat shockprotein (hsp) 70 promoter, Autographa californica nuclear polyhedrosisvirus immediate-early gene promoter (ie-1) and the delayed early 39Kpromoter, baculovirus p10 promoter, and the Drosophila metallothioneinpromoter. A second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the complement C1s inhibitorpolypeptide into a baculovirus genome maintained in E. coli as a largeplasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). In addition,transfer vectors can include an in-frame fusion with DNA encoding anepitope tag at the C- or N-terminus of the expressed complement C1sinhibitor polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyeret al., Proc. Nat'l Acad Sci. 82:7952 (1985)). Using a technique knownin the art, a transfer vector containing a complement C1s inhibitorpeptide or polypeptide encoding sequence is transformed into E. coli,and screened for bacmids, which contain an interrupted lacZ geneindicative of recombinant baculovirus. The bacmid DNA containing therecombinant baculovirus genome is then isolated using common techniques.

[0121] The illustrative PFASTBAC vector can be modified to aconsiderable degree. For example, the polyhedrin promoter can be removedand substituted with the baculovirus basic protein promoter (also knownas Pcor, p6.9 or MP promoter) which is expressed earlier in thebaculovirus infection, and has been shown to be advantageous forexpressing secreted proteins (see, for example, Hill-Perkins and Possee,J. Gen. Virol. 71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551(1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). Insuch transfer vector constructs, a short or long version of the basicprotein promoter can be used. Moreover, transfer vectors can beconstructed with secretory signal sequences derived from insectproteins. For example, a secretory signal sequence from EcdysteroidGlucosyltransferase (EGT), honey bee Melittin (Invitrogen Corporation;Carlsbad, Calif.), or baculovirus gp67 (PharMingen: San Diego, Calif.)can be used in constructs to express the C1s inhibitory peptide orpolypeptide.

[0122] The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for theT. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

[0123] Established techniques for producing recombinant proteins inbaculovirus systems are provided by Bailey et al., “Manipulation ofBaculovirus Vectors,” in Methods in Molecular Biology, Volume 7: GeneTransfer and Expression Protocols, Murray (ed.), pages 147-168 (TheHumana Press, Inc. 1991), by Patel et al., “The baculovirus expressionsystem,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover etal. (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel(1995) at pages 16-37 to 16-57, by Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995), and by Lucknow,“Insect Cell Expression Technology,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), pages 183-218 (John Wiley & Sons,Inc. 1996).

[0124] Fungal cells, including yeast cells, can also be used to expressthe genes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsmanet al., U.S. Pat. No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092)and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

[0125] Transformation systems for other yeasts, including Hansenulapolymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichiamethanolica, Pichia guillermondii and Candida maltosa are known in theart. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459(1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may beutilized according to the methods of McKnight et al., U.S. Pat. No.4,935,349. Methods for transforming Acremonium chrysogenum are disclosedby Sumino et al., U.S. Pat. No. 5,162,228. Methods for transformingNeurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.

[0126] For example, the use of Pichia methanolica as host for theproduction of recombinant proteins is disclosed by Raymond, U.S. Pat.No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast14:11-23 (1998), and in international publication Nos. WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which can be linearized prior totransformation. For polypeptide production in P. methanolica thepromoter and terminator in the plasmid can be that of a P. methanolicagene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2).Other useful promoters include those of the dihydroxyacetone synthase(DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. Tofacilitate integration of the DNA into the host chromosome, the entireexpression segment of the plasmid can be flanked at both ends by hostDNA sequences. A suitable selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells deficient in vacuolar protease genes (PEP4and PRB1) can be used. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

[0127] Expression vectors can also be introduced into plant protoplasts,intact plant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al, “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

[0128] Alternatively, nucleotide sequence encoding complement C1sinhibitor peptides and polypeptides can be expressed in prokaryotic hostcells. Suitable promoters that can be used to express eukaryoticpolypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the P_(R) and P_(L) promoters of bacteriophage lambda,the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZpromoters of E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloramphenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al. (1995).

[0129] Illustrative prokaryotic hosts include E. coli and Bacillussubtilus. Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DHI, DH4I, DH5, DH5I, DH5IEF′, DH5IMCR, DHIOB, DH10B/p3,DHlS, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, M1120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

[0130] When expressing a complement C1s inhibitor peptide or polypeptidein bacteria such as E. coli, the polypeptide may be retained in thecytoplasm, typically as insoluble granules, or may be directed to theperiplasmic space by a bacterial secretion sequence. In the former case,the cells are lysed, and the granules are recovered and denatured using,for example, guanidine isothiocyanate or urea. The denatured polypeptidecan then be refolded and dimerized by diluting the denaturant, such asby dialysis against a solution of urea and a combination of reduced andoxidized glutathione, followed by dialysis against a buffered salinesolution. In the latter case, the polypeptide can be recovered from theperiplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and recovering the protein, therebyobviating the need for denaturation and refolding.

[0131] Methods for expressing proteins in prokaryotic hosts arewell-known to those of skill in the art (see, for example, Williams etal., “Expression of foreign proteins in E. coli using plasmid vectorsand purification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

[0132] Standard methods for introducing expression vectors intobacterial, yeast, insect, and plant cells are provided, for example, byAusubel (1995).

[0133] General methods for expressing and recovering foreign proteinproduced by a mammalian cell system are provided by, for example,Etcheverry, “Expression of Engineered Proteins in Mammalian CellCulture,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 163 (Wiley-Liss, Inc. 1996). Standard techniques forrecovering protein produced by a bacterial system is provided by, forexample, Grisshammer et al., “Purification of over-produced proteinsfrom E. coli cells,” in DNA Cloning 2: Expression Systems, 2nd Edition,Glover et al. (eds.), pages 59-92 (Oxford University Press 1995).Established methods for isolating recombinant proteins from abaculovirus system are described by Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995).

[0134] 6. Isolation of Complement C1s Inhibitor Polypeptides

[0135] The peptides and polypeptides of the present invention can bepurified to at least about 80% purity, to at least about 90% purity, toat least about 95% purity, or even greater than 95% purity with respectto contaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. The peptides andpolypeptides of the present invention may also be purified to apharmaceutically pure state, which is greater than 99.9% pure. Incertain preparations, a purified polypeptide is substantially free ofother polypeptides, particularly other polypeptides of animal origin.

[0136] Fractionation and/or conventional purification methods can beused to obtain preparations of complement C1s inhibitor peptides andpolypeptides purified from recombinant host cells. Numerous methods forpurifying proteins are known in the art. In general, ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties.

[0137] Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Selection of a particular method for polypeptideisolation and purification is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods (Pharmacia LKBBiotechnology 1988), and Doonan, Protein Purification Protocols (TheHumana Press 1996).

[0138] The peptides and polypeptides of the present invention can alsobe isolated by exploitation of particular properties. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins, including those comprising polyhistidinetags. Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

[0139] Complement C1s inhibitor polypeptides or fragments thereof may beglycosylated or non-glycosylated, pegylated or non-pegylated, and may ormay not include an initial methionine amino acid residue.

[0140] 7. Complement C1s Inhibitor Polypeptide-polymer Conjugates

[0141] The peptides and polypeptides of the present invention can beprepared as conjugates with various polymers. For example, such polymercan be water soluble so that the complement C1s inhibitor conjugate doesnot precipitate in an aqueous environment, such as a physiologicalenvironment. An example of a suitable polymer is one that has beenmodified to have a single reactive group, such as an active ester foracylation, or an aldehyde for alkylation, In this way, the degree ofpolymerization can be controlled. An example of a reactive aldehyde ispolyethylene glycol propionaldehyde, or mono-(C₁-C₁₀) alkoxy, or aryloxyderivatives thereof (see, for example, Harris, et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Moreover, amixture of polymers can be used to produce complement C1s inhibitorconjugates.

[0142] Complement C1s inhibitor conjugates used for therapy can comprisepharmaceutically acceptable water-soluble polymer moieties. Suitablewater-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C₁-C₁₀)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. Å complement C1s inhibitor peptide or polypeptideconjugate can also comprise a mixture of such water-soluble polymers.

[0143] One example of a complement C1s inhibitor peptide or polypeptideconjugate comprises a complement C1s inhibitor moiety and a polyalkyloxide moiety attached to the N-terminus of the complement C1s inhibitormoiety. PEG is one suitable polyalkyl oxide. As an illustration, acomplement C1s inhibitor peptide or polypeptide can be modified withPEG, a process known as “PEGylation.” PEGylation of peptides andpolypeptides can be carried out by any of the PEGylation reactions knownin the art (see, for example, EP 0 154 316, Delgado et al., CriticalReviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan andSpreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int JHematol 68:1 (1998)). For example, PEGylation can be performed by anacylation reaction or by an alkylation reaction with a reactivepolyethylene glycol molecule. In an alternative approach, complement C1sinhibitor peptide and polypeptide conjugates are formed by condensingactivated PEG, in which a terminal hydroxy or amino group of PEG hasbeen replaced by an activated linker (see, for example, Karasiewicz etal., U.S. Pat. No. 5,382,657).

[0144] PEGylation by acylation typically requires reacting an activeester derivative of PEG with a complement C1s inhibitor peptide orpolypeptide. An example of an activated PEG ester is PEG esterified toN-hydroxysuccinimide. As used herein, the term “acylation” includes thefollowing types of linkages between a complement C1s inhibitor peptideor polypeptide and a water soluble polymer: amide, carbamate, urethane,and the like. Methods for preparing PEGylated complement C1s inhibitorpeptides or polypeptides by acylation will typically comprise the stepsof (a) reacting a complement C1s inhibitor moiety with PEG (such as areactive ester of an aldehyde derivative of PEG) under conditionswhereby one or more PEG groups attach to the complement C1s inhibitormoiety, and (b) obtaining the reaction product(s). Generally, theoptimal reaction conditions for acylation reactions will be determinedbased upon known parameters and desired results. For example, the largerthe ratio of PEG: complement C1s inhibitor moiety, the greater thepercentage of polyPEGylated complement C1s inhibitor product.

[0145] The product of PEGylation by acylation is typically apolyPEGylated complement C1s inhibitor product, wherein the lysineε-amino groups are PEGylated via an acyl linking group. An example of aconnecting linkage is an amide. Typically, the resulting complement C1sinhibitor moiety will be at least 95% mono-, di-, or tri-pegylated,although some species with higher degrees of PEGylation may be formeddepending upon the reaction conditions. PEGylated species can beseparated from unconjugated complement C1s inhibitor peptides andpolypeptides using standard purification methods, such as dialysis,ultrafiltration, ion exchange chromatography, affinity chromatography,and the like.

[0146] PEGylation by alkylation generally involves reacting a terminalaldehyde derivative of PEG with a complement C1s inhibitor moiety in thepresence of a reducing agent. PEG groups can be attached to thepolypeptide via a —CH₂—NH group.

[0147] Derivatization via reductive alkylation to produce amonoPEGylated product takes advantage of the differential reactivity ofdifferent types of primary amino groups available for derivatization.Typically, the reaction is performed at a pH that allows one to takeadvantage of the pKa differences between the α-amino groups of thelysine residues and the α-amino group of the N-terminal residue of theprotein. By such selective derivatization, attachment of a water-solublepolymer that contains a reactive group such as an aldehyde, to a proteinis controlled. The conjugation with the polymer occurs predominantly atthe N-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of complementC1s inhibitor monopolymer conjugates.

[0148] Reductive alkylation to produce a substantially homogenouspopulation of monopolymer complement C1s inhibitor conjugate moleculecan comprise the steps of: (a) reacting a complement C1s inhibitorpeptide or polypeptide with a reactive PEG under reductive alkylationconditions at a pH suitable to permit selective modification of theα-amino group at the amino terminus of the complement C1s inhibitormoiety, and (b) obtaining the reaction product(s). The reducing agentused for reductive alkylation should be stable in aqueous solution andable to reduce only the Schiff base formed in the initial process ofreductive alkylation. Illustrative reducing agents include sodiumborohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

[0149] For a substantially homogenous population of monopolymercomplement C1s inhibitor conjugates, the reductive alkylation reactionconditions are those which permit the selective attachment of the watersoluble polymer moiety to the N-terminus of the complement C1s inhibitormoiety. Such reaction conditions generally provide for pKa differencesbetween the lysine amino groups and the α-amino group at the N-terminus.The pH also affects the ratio of polymer to protein to be used. Ingeneral, if the pH is lower, a larger excess of polymer to protein willbe desired because the less reactive the N-terminal α-group, the morepolymer is needed to achieve optimal conditions. If the pH is higher,the polymer: complement C1s inhibitor moiety need not be as largebecause more reactive groups are available. Typically, the pH will fallwithin the range of 3 to 9, or 3 to 6.

[0150] General methods for producing conjugates comprising a polypeptideand water-soluble polymer moieties are known in the art. See, forexample, Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al.,U.S. Pat. No. 5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636(1996), Monkarsh et al., Anal. Biochem. 247:434 (1997)).

[0151] The present invention contemplates compositions comprising apeptide or polypeptide described herein. Such compositions can furthercomprise a carrier. The carrier can be a conventional organic orinorganic carrier. Examples of carriers include water, buffer solution,alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.

[0152] 8. Therapeutic Uses of Complement C1s Inhibitor Peptides andPolypeptides

[0153] The present invention includes the use of complement C1sinhibitory peptide and polypeptides for therapy in mammals. As anillustration, these molecules can be used to treat systemic lupuserythematosus, rheumatoid arthritis, serum sickness, various hemolyticanemias, myasthenia gravis, and certain forms of nephritis. Inhibitionof complement activation can also be used when tissue damage is causedby vascular injury, such as myocardial infarction, cerebral vascularaccidents, reperfusion of ischemic tissue, and acute shock lungsyndrome. Complement activation inhibitors are also useful fordecreasing the rejection of transplanted tissues. For example,inhibition of complement can decrease vascular leak syndrome followingbone marrow transplantation, or lung transplantation. The effect ofvascular leak syndrome can also be decreased following open heartsurgery. The complement inhibitors described herein can also be used toreduce mortality associated with the occurrence of severe thermal injuryand septic shock. Additional therapeutic uses of complement inhibitorsare recognized by those of skill in the art.

[0154] The present invention contemplates both veterinary and humantherapeutic uses. Illustrative subjects include mammalian subjects, suchas farm animals, domestic animals, and human patients.

[0155] Generally, the dosage of administered polypeptide or peptide willvary depending upon such factors as the subject's age, weight, height,sex, general medical condition and previous medical history. Typically,it is desirable to provide the recipient with a dosage of a complementC1s inhibitor peptide or polypeptide, which is in the range of fromabout 1 pg/kg to 10 mg/kg (amount of agent/body weight of subject),although a lower or higher dosage also may be administered ascircumstances dictate.

[0156] Administration of a complement C1s inhibitor peptide orpolypeptide to a subject can be intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic proteins by injection, theadministration may be by continuous infusion or by single or multipleboluses.

[0157] Additional routes of administration include oral,mucosal-membrane, pulmonary, and transcutaneous. Oral delivery issuitable for polyester microspheres, zein nicrospheres, proteinoidmicrospheres, polycyanoacrylate microspheres, and lipid-based systems(see, for example, DiBase and Morrel, “Oral Delivery ofMicroencapsulated Proteins,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). Thefeasibility of an intranasal delivery is exemplified by such a mode ofinsulin administration (see, for example, Hinchcliffe and Illum, Adv.Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles comprisingcomplement C1s inhibitor peptides or polypeptides can be prepared andinhaled with the aid of dry-powder dispersers, liquid aerosolgenerators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343(1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)). Thisapproach is illustrated by the AERX diabetes management system, which isa hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al., Science 269:850(1995)). Transdermal delivery using electroporation provides anothermeans to administer complement C1s inhibitor peptides or polypeptides(Potts et al., Pharm. Biotechnol. 10:213 (1997)).

[0158] A pharmaceutical composition comprising a complement C1sinhibitor peptide or polypeptide can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thetherapeutic proteins are combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient subject. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

[0159] For purposes of therapy, a complement C1s inhibitor peptide orpolypeptide and a pharmaceutically acceptable carrier are administeredto a subject in a therapeutically effective amount. A combination of acomplement C1s inhibitor peptide, or polypeptide, and a pharmaceuticallyacceptable carrier is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient subject.

[0160] A pharmaceutical composition comprising a complement C1sinhibitor peptide or polypeptide can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).

[0161] Liposomes provide one means to deliver therapeutic polypeptidesto a subject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

[0162] Liposomes can adsorb to virtually any type of cell and thenslowly release the encapsulated agent. Alternatively, an absorbedliposome may be endocytosed by cells that are phagocytic. Endocytosis isfollowed by intralysosomal degradation of liposomal lipids and releaseof the encapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci.446:368 (1985)). After intravenous administration, small liposomes (0.1to 1.0 μm) are typically taken up by cells of the reticuloendothelialsystem, located principally in the liver and spleen, whereas liposomeslarger than 3.0 μm are deposited in the lung. This preferential uptakeof smaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

[0163] The reticuloendothelial system can be circumvented by severalmethods including saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

[0164] Liposomes can also be prepared to target particular cells ororgans by varying phospholipid composition or by inserting receptors orligands into the liposomes. For example, liposomes, prepared with a highcontent of a nonionic surfactant, have been used to target the liver(Hayakawa et al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm.Bull 16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

[0165] Alternatively, various targeting ligands can be bound to thesurface of the liposome, such as antibodies, antibody fragments,carbohydrates, vitamins, and transport proteins. For example, liposomescan be modified with branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull.20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

[0166] In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

[0167] A complement C1s inhibitor peptide or polypeptide can beencapsulated within liposomes using standard techniques of proteinmicroencapsulation (see, for example, Anderson et al., Infect. Immun.31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990), and Cohenet al., Biochim. Biophys. Acta 1063:95 (1991), Alving et al.“Preparation and Use of Liposomes in Immunological Studies,” in LiposomeTechnology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRCPress 1993), Wassef et al., Meth. Enzymol. 149:124 (1987)). As notedabove, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta 1150:9(1993)).

[0168] Degradable polymer microspheres have been designed to maintainhigh systemic levels of therapeutic proteins. Microspheres are preparedfrom degradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

[0169] Other dosage forms can be devised by those skilled in the art, asshown, for example, by Ansel and Popovich, Pharmaceutical Dosage Formsand Drug Delivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro(ed.), Remington's Pharmaceutical Sciences, 19^(th) Edition (MackPublishing Company 1995), and by Ranade and Hollinger, Drug DeliverySystems (CRC Press 1996).

[0170] As an illustration, pharmaceutical compositions may be suppliedas a kit comprising a container that comprises a complement C1sinhibitor peptide or polypeptide. Therapeutic polypeptides can beprovided in the form of an injectable solution for single or multipledoses, or as a sterile powder that will be reconstituted beforeinjection. Alternatively, such a kit can include a dry-powder disperser,liquid aerosol generator, or nebulizer for administration of atherapeutic polypeptide. Such a kit may further comprise writteninformation on indications and usage of the pharmaceutical composition.Moreover, such information may include a statement that the complementC1s inhibitor peptide or polypeptide is contraindicated in patients withknown hypersensitivity to complement C1s inhibitor peptides orpolypeptides.

[0171] A variety of animal models are available to examine the efficacyof particular formulations of the inhibitory peptides and polypeptidesdescribed herein. For example, animal models provide a means to testefficacy in the treatment of sepsis, pulmonary dysfunction,pancreatitis, acute myocardial infarction, lung transplantation, trauma,thermal injury, and the like (for a review, see Caliezi et al.,Pharmacol. Rev. 52:91 (2000)).

[0172] The present invention, thus generally described, will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention.

EXAMPLE 1 C1s Enzyme Assay and Classical Complement Hemolysis Assay

[0173] C1s enzyme assays were performed in 96 well plates preincubatedin the assay buffer for 30 minutes at room temperature. Activated humanC1s (Calbiochem-Novabiochem Corporation; San Diego, Calif.); final assayconcentration: 1.25 μg/ml), was incubated with samples in 50 mM Tris-HCl(pH 8) that contained 116 mM sodium chloride and 0.05% Polysorbate 80.The chromogenic substrate, Pefa-C1E (Centerchem, Inc.; Stamford, Conn.;final assay concentration: 0.4 mM), was added to the assay wells and theplate was read at 405 nm, 37° C., for 30 minutes at 20 second intervalson a SPECTRAmax PLUS plate reader (Molecular Devices Corporation;Sunnyvale, Calif.). Inhibition was determined as a decrease in Vmax,which is calculated as the maximum A milli-absorbance units/min over theassay period.

[0174] An assay was performed to determine the effect of BD001 onclassical complement hemolysis. A pool of pre-sensitized sheeperythrocytes (DiaMedix Corporation; Miami, Fla.) was distributed into 50milliliter conical tubes, and centrifuged at 1000 rpm for 10 minutes.Supematants were discarded, and remaining pellets were resuspended inGelatin Veronal buffer (Sigma Chemical Company; St. Louis, Mo.). Thepellets were washed twice again with Gelatin Veronal buffer.

[0175] A standard curve of lysed cells was created by diluting cells indistilled water. The cells were resuspended in an appropriate volume toprovide a 100% lysis absorbance reading of 0.7 AU at 415 nm. A serumserial dilution was performed to determine which serum dilution willprovide 75% lysis; this is the serum dilution that was used for theassay. The assay was performed by incubating: 100 μl Gelatin Veronalbuffer, 50 μl of a serum dilution or Gelatin Veronal buffer; 50 μl ofsample or Gelatin Veronal buffer; and 50 μl of pre-sensitized rabbiterythrocytes for one hour at 37° C. After centrifuging the plate for 10minutes at 1500 rpm, 100 μl of supernatant were transferred to a newplate. The plates were read at 415 nm on a SPECTRAmax PLUS plate reader.Inhibition was measured as a decrease in absorbance from maximum lysis.

[0176] Differences were observed in the activity of BD001 proteinisolated from baculovirus and Pichia systems. According to the C1senzyme assay, the IC₅₀ calculated for the baculovirus material was 5.2nM, whereas the Pichia material provided an IC₅₀ of 44 nM, or abouteight-fold less active than the baculovirus material.

EXAMPLE 2 Effects of Posttranslational Modification on BD001 Activity

[0177] Three types of post-translational modifications have beenidentified in BDO01: (1) glycosylation at Asn²³ consisting of afucosylated complex-type core; (2) sulfation of at least one of Tyr¹¹⁷,Tyr¹¹⁹, and Tyr¹²¹; and (3) proteolytic cleavage after Arg⁶⁵. The firsttwo modifications have been identified in native BD001 as well asrecombinant BD001 expressed in Baculovirus. The third modification isseen in a fraction of the recombinant BD001 expressed in Baculovirus. Toevaluate the effect of these posttranslational modifications onactivity, sulfate groups were removed from recombinant BD001 using arylsulfatase (Sigma Chemical Co.; St. Louis, Mo.) in 10 mM acetate, 120 mMNaCl (pH 5.5), recombinant BD001 proteins were deglycosylated usingPNGase F (CALBIOCHEM-NOVABIOCHEM Corp.; La Jolla, Calif.) in buffersupplied by the manufacturer, and the cleaved recombinant BD001 specieswas separated from intact BD001 using reverse phase high-pressure liquidchromatography (Zorbax 300SB-C18, 10-30% ACN gradient, 60° C.).Following treatment, the proteins were lyophilized, and thenreconstituted in MilliQ water. The identity of each species was verifiedusing mass spectroscopy, and protein concentrations were measured usingabsorbance at 280 nm. The C1s enzyme assay, described above, was used toevaluate activity.

[0178] As shown in Table 6, removal of the glycosylation had anegligible effect on the IC₅₀ of recombinant BD001, whereas the removalof sulfate had a significant effect on the IC₅₀ of recombinant BD001. Inaddition, cleaved BD001 was found to have an approximate 10 fold higherIC₅₀ than the matched, intact form. TABLE 6 Protein IC₅₀ (nM) BD001isolated from tissue 4.0,4.7 Recombinant BD001 2.5 Deglycosylatedrecombinant BD001 2.86 Desulfated recombinant BD001 36 Cleavedrecombinant BD001 19.8 Deglycosylated and cleaved recombinant BD001 21.9Desulfated and cleaved recombinant BD001 558

[0179] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 140 1 122 PRT Haementaria ghilianii 1 Ala Lys Lys Lys Leu Pro Lys CysGln Lys Gln Glu Asp Cys Gly Ser 1 5 10 15 Trp Asp Leu Lys Cys Asn AsnVal Thr Lys Lys Cys Glu Cys Arg Asn 20 25 30 Gln Val Cys Gly Arg Gly CysPro Lys Glu Arg Tyr Gln Arg Asp Lys 35 40 45 Tyr Gly Cys Arg Lys Cys LeuCys Lys Gly Cys Asp Gly Phe Lys Cys 50 55 60 Arg Leu Gly Cys Thr Tyr GlyPhe Lys Thr Asp Lys Lys Gly Cys Glu 65 70 75 80 Ala Phe Cys Thr Cys AsnThr Lys Glu Thr Ala Cys Val Asn Ile Trp 85 90 95 Cys Thr Asp Pro Tyr LysCys Asn Pro Glu Ser Gly Arg Cys Glu Asp 100 105 110 Pro Asn Glu Glu TyrGlu Tyr Asp Tyr Glu 115 120 2 10 PRT Artificial Sequence C1s exositebinding moiety 2 Pro Asn Glu Glu Tyr Glu Tyr Asp Tyr Glu 1 5 10 3 10 PRTArtificial Sequence C1s exosite binding moiety 3 Pro Asn Glu Glu Xaa GluTyr Asp Tyr Glu 1 5 10 4 10 PRT Artificial Sequence C1s exosite bindingmoiety 4 Pro Asn Glu Glu Tyr Glu Xaa Asp Tyr Glu 1 5 10 5 10 PRTArtificial Sequence C1s exosite binding moiety 5 Pro Asn Glu Glu Tyr GluTyr Asp Xaa Glu 1 5 10 6 10 PRT Artificial Sequence C1s exosite bindingmoiety 6 Pro Asn Glu Glu Xaa Glu Xaa Asp Tyr Glu 1 5 10 7 10 PRTArtificial Sequence C1s exosite binding moiety 7 Pro Asn Glu Glu Xaa GluTyr Asp Xaa Glu 1 5 10 8 10 PRT Artificial Sequence C1s exosite bindingmoiety 8 Pro Asn Glu Glu Tyr Glu Xaa Asp Xaa Glu 1 5 10 9 10 PRTArtificial Sequence C1s exosite binding moiety 9 Pro Asn Glu Glu Xaa GluXaa Asp Xaa Glu 1 5 10 10 10 PRT Artificial Sequence C1s exosite bindingmoiety 10 Pro Asn Glu Glu Xaa Glu Tyr Asp Tyr Glu 1 5 10 11 10 PRTArtificial Sequence C1s exosite binding moiety 11 Pro Asn Glu Glu TyrGlu Xaa Asp Tyr Glu 1 5 10 12 10 PRT Artificial Sequence C1s exositebinding moiety 12 Pro Asn Glu Glu Tyr Glu Tyr Asp Xaa Glu 1 5 10 13 10PRT Artificial Sequence C1s exosite binding moiety 13 Pro Asn Glu GluXaa Glu Xaa Asp Tyr Glu 1 5 10 14 10 PRT Artificial Sequence C1s exositebinding moiety 14 Pro Asn Glu Glu Xaa Glu Tyr Asp Xaa Glu 1 5 10 15 10PRT Artificial Sequence C1s exosite binding moiety 15 Pro Asn Glu GluTyr Glu Xaa Asp Xaa Glu 1 5 10 16 10 PRT Artificial Sequence C1s exositebinding moiety 16 Pro Asn Glu Glu Xaa Glu Xaa Asp Xaa Glu 1 5 10 17 10PRT Artificial Sequence C1s exosite binding moiety 17 Pro Asn Glu GluXaa Glu Xaa Asp Tyr Glu 1 5 10 18 10 PRT Artificial Sequence C1s exositebinding moiety 18 Pro Asn Glu Glu Xaa Glu Xaa Asp Tyr Glu 1 5 10 19 10PRT Artificial Sequence C1s exosite binding moiety 19 Pro Asn Glu GluXaa Glu Tyr Asp Xaa Glu 1 5 10 20 10 PRT Artificial Sequence C1s exositebinding moiety 20 Pro Asn Glu Glu Xaa Glu Tyr Asp Xaa Glu 1 5 10 21 10PRT Artificial Sequence C1s exosite binding moiety 21 Pro Asn Glu GluTyr Glu Xaa Asp Xaa Glu 1 5 10 22 10 PRT Artificial Sequence C1s exositebinding moiety 22 Pro Asn Glu Glu Tyr Glu Xaa Asp Xaa Glu 1 5 10 23 10PRT Artificial Sequence C1s exosite binding moiety 23 Pro Asn Glu GluXaa Glu Xaa Asp Xaa Glu 1 5 10 24 10 PRT Artificial Sequence C1s exositebinding moiety 24 Pro Asn Glu Glu Xaa Glu Xaa Asp Xaa Glu 1 5 10 25 10PRT Artificial Sequence C1s exosite binding moiety 25 Pro Asn Glu GluXaa Glu Xaa Asp Xaa Glu 1 5 10 26 10 PRT Artificial Sequence C1s exositebinding moiety 26 Pro Asn Glu Glu Xaa Glu Xaa Asp Xaa Glu 1 5 10 27 10PRT Artificial Sequence C1s exosite binding moiety 27 Pro Asn Glu GluXaa Glu Xaa Asp Xaa Glu 1 5 10 28 11 PRT Artificial Sequence C1s exositebinding moiety 28 Ala Asn Glu Asp Xaa Glu Asp Tyr Glu Tyr Asp 1 5 10 2911 PRT Artificial Sequence C1s exosite binding moiety 29 Ala Asn Glu AspTyr Glu Asp Xaa Glu Tyr Asp 1 5 10 30 11 PRT Artificial Sequence C1sexosite binding moiety 30 Ala Asn Glu Asp Tyr Glu Asp Tyr Glu Xaa Asp 15 10 31 11 PRT Artificial Sequence C1s exosite binding moiety 31 Ala AsnGlu Asp Xaa Glu Asp Xaa Glu Tyr Asp 1 5 10 32 11 PRT Artificial SequenceC1s exosite binding moiety 32 Ala Asn Glu Asp Xaa Glu Asp Tyr Glu XaaAsp 1 5 10 33 11 PRT Artificial Sequence C1s exosite binding moiety 33Ala Asn Glu Asp Tyr Glu Asp Xaa Glu Xaa Asp 1 5 10 34 11 PRT ArtificialSequence C1s exosite binding moiety 34 Ala Asn Glu Asp Xaa Glu Asp XaaGlu Xaa Asp 1 5 10 35 11 PRT Artificial Sequence C1s exosite bindingmoiety 35 Ala Asn Glu Asp Xaa Glu Asp Tyr Glu Tyr Asp 1 5 10 36 11 PRTArtificial Sequence C1s exosite binding moiety 36 Ala Asn Glu Asp TyrGlu Asp Xaa Glu Tyr Asp 1 5 10 37 11 PRT Artificial Sequence C1s exositebinding moiety 37 Ala Asn Glu Asp Tyr Glu Asp Tyr Glu Xaa Asp 1 5 10 3811 PRT Artificial Sequence C1s exosite binding moiety 38 Ala Asn Glu AspXaa Glu Asp Xaa Glu Tyr Asp 1 5 10 39 11 PRT Artificial Sequence C1sexosite binding moiety 39 Ala Asn Glu Asp Xaa Glu Asp Tyr Glu Xaa Asp 15 10 40 11 PRT Artificial Sequence C1s exosite binding moiety 40 Ala AsnGlu Asp Tyr Glu Asp Xaa Glu Xaa Asp 1 5 10 41 11 PRT Artificial SequenceC1s exosite binding moiety 41 Ala Asn Glu Asp Xaa Glu Asp Xaa Glu XaaAsp 1 5 10 42 11 PRT Artificial Sequence C1s exosite binding moiety 42Ala Asn Glu Asp Xaa Glu Asp Xaa Glu Tyr Asp 1 5 10 43 11 PRT ArtificialSequence C1s exosite binding moiety 43 Ala Asn Glu Asp Xaa Glu Asp XaaGlu Tyr Asp 1 5 10 44 11 PRT Artificial Sequence C1s exosite bindingmoiety 44 Ala Asn Glu Asp Xaa Glu Asp Tyr Glu Xaa Asp 1 5 10 45 11 PRTArtificial Sequence C1s exosite binding moiety 45 Ala Asn Glu Asp XaaGlu Asp Tyr Glu Xaa Asp 1 5 10 46 11 PRT Artificial Sequence C1s exositebinding moiety 46 Ala Asn Glu Asp Tyr Glu Asp Xaa Glu Xaa Asp 1 5 10 4711 PRT Artificial Sequence C1s exosite binding moiety 47 Ala Asn Glu AspTyr Glu Asp Xaa Glu Xaa Asp 1 5 10 48 11 PRT Artificial Sequence C1sexosite binding moiety 48 Ala Asn Glu Asp Xaa Glu Asp Xaa Glu Xaa Asp 15 10 49 11 PRT Artificial Sequence C1s exosite binding moiety 49 Ala AsnGlu Asp Xaa Glu Asp Xaa Glu Xaa Asp 1 5 10 50 11 PRT Artificial SequenceC1s exosite binding moiety 50 Ala Asn Glu Asp Xaa Glu Asp Xaa Glu XaaAsp 1 5 10 51 11 PRT Artificial Sequence C1s exosite binding moiety 51Ala Asn Glu Asp Xaa Glu Asp Xaa Glu Xaa Asp 1 5 10 52 11 PRT ArtificialSequence C1s exosite binding moiety 52 Ala Asn Glu Asp Xaa Glu Asp XaaGlu Xaa Asp 1 5 10 53 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 53 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 54 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 54 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrSer Asn Thr 20 25 30 55 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 55 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 56 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 56 Gly Ser Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 57 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 57 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 58 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 58 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 59 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 59 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 60 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 60 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 61 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 61 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 62 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 62 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 63 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 63 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 64 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 64 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 65 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 65 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 66 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 66 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrSer Asn Thr 20 25 30 67 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 67 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 68 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 68 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 69 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 69 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrSer Asn Thr 20 25 30 70 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 70 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrSer Asn Thr 20 25 30 71 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 71 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 72 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 72 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 73 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 73 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 74 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 74 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 75 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 75 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 76 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 76 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 77 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 77 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 78 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 78 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 79 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 79 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 80 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 80 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 81 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 81 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 82 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 82 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 83 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 83 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 84 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 84 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Ser ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrSer Asn Thr 20 25 30 85 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 85 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 86 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 86 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 87 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 87 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 88 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 88 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 89 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 89 Gly Ser Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 90 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 90 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 91 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 91 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 92 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 92 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 93 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 93 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 94 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 94 Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 95 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 95 Gly Ser Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Cys ThrCys Asn Thr 20 25 30 96 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 96 Gly Ser Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Cys Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 97 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 97 Gly Cys Asp Gly Phe Lys Ser Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 98 30 PRT Artificial Sequence C1s catalyticsite-directed moiety 98 Gly Ser Asp Gly Phe Lys Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys 1 5 10 15 Thr Asp Lys Lys Gly Ser Glu Ala Phe Ser ThrCys Asn Thr 20 25 30 99 6 PRT Artificial Sequence C1s catalyticsite-directed moiety 99 Cys Arg Leu Gly Cys Thr 1 5 100 7 PRT ArtificialSequence C1s catalytic site-directed moiety 100 Cys Arg Leu Gly Cys ThrTyr 1 5 101 8 PRT Artificial Sequence C1s catalytic site-directed moiety101 Cys Arg Leu Gly Cys Thr Tyr Gly 1 5 102 9 PRT Artificial SequenceC1s catalytic site-directed moiety 102 Cys Arg Leu Gly Cys Thr Tyr GlyPhe 1 5 103 10 PRT Artificial Sequence C1s catalytic site-directedmoiety 103 Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys 1 5 10 104 11 PRTArtificial Sequence C1s catalytic site-directed moiety 104 Cys Arg LeuGly Cys Thr Tyr Gly Phe Lys Thr 1 5 10 105 12 PRT Artificial SequenceC1s catalytic site-directed moiety 105 Cys Arg Leu Gly Cys Thr Tyr GlyPhe Lys Thr Asp 1 5 10 106 13 PRT Artificial Sequence C1s catalyticsite-directed moiety 106 Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr AspLys 1 5 10 107 14 PRT Artificial Sequence C1s catalytic site-directedmoiety 107 Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys 1 510 108 15 PRT Artificial Sequence C1s catalytic site-directed moiety 108Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly 1 5 10 15109 16 PRT Artificial Sequence C1s catalytic site-directed moiety 109Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly Cys 1 5 1015 110 17 PRT Artificial Sequence C1s catalytic site-directed moiety 110Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly Cys 1 5 1015 Glu 111 18 PRT Artificial Sequence C1s catalytic site-directed moiety111 Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly Cys 1 510 15 Glu Ala 112 19 PRT Artificial Sequence C1s catalytic site-directedmoiety 112 Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys GlyCys 1 5 10 15 Glu Ala Phe 113 20 PRT Artificial Sequence C1s catalyticsite-directed moiety 113 Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr AspLys Lys Gly Cys 1 5 10 15 Glu Ala Phe Cys 20 114 21 PRT ArtificialSequence C1s catalytic site-directed moiety 114 Cys Arg Leu Gly Cys ThrTyr Gly Phe Lys Thr Asp Lys Lys Gly Cys 1 5 10 15 Glu Ala Phe Cys Thr 20115 22 PRT Artificial Sequence C1s catalytic site-directed moiety 115Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly Cys 1 5 1015 Glu Ala Phe Cys Thr Cys 20 116 23 PRT Artificial Sequence C1scatalytic site-directed moiety 116 Cys Arg Leu Gly Cys Thr Tyr Gly PheLys Thr Asp Lys Lys Gly Cys 1 5 10 15 Glu Ala Phe Cys Thr Cys Asn 20 1176 PRT Artificial Sequence C1s catalytic site-directed moiety 117 Leu GlnArg Ala Leu Glu 1 5 118 24 PRT Artificial Sequence C1s catalyticsite-directed moiety 118 Leu Gln Arg Ala Leu Glu Ile Leu Pro Asn Arg ValThr Ile Lys Ala 1 5 10 15 Asn Arg Pro Phe Leu Val Phe Ile 20 119 10 PRTArtificial Sequence C1s exosite binding moiety 119 Asn Glu Asp Tyr GluAsp Tyr Glu Tyr Asp 1 5 10 120 23 PRT Artificial Sequence Polypeptidelinker 120 Lys Glu Thr Ala Cys Val Asn Ile Trp Cys Thr Asp Pro Tyr LysCys 1 5 10 15 Asn Pro Glu Ser Gly Arg Cys 20 121 29 PRT ArtificialSequence C1s catalytic site-directed moiety 121 Cys Asp Gly Phe Lys CysArg Leu Gly Cys Thr Tyr Gly Phe Lys Thr 1 5 10 15 Asp Lys Lys Gly CysGlu Ala Phe Cys Thr Cys Asn Thr 20 25 122 4 PRT Artificial SequencePeptide linker 122 Ala Leu Xaa Xaa 1 123 25 PRT Artificial SequencePolypeptide linker 123 Lys Glu Thr Ala Cys Val Asn Ile Trp Cys Thr AspPro Tyr Lys Cys 1 5 10 15 Asn Pro Glu Ser Gly Arg Cys Glu Asp 20 25 1245 PRT Artificial Sequence Peptide linker 124 Ala Leu Xaa Xaa Cys 1 5 12510 PRT Artificial Sequence Complement C1s inhibitor 125 Pro Asn Glu GluTyr Glu Tyr Glu Tyr Glu 1 5 10 126 21 PRT Artificial Sequence Formula ofa complement C1s inhibitor 126 Xaa Asn Xaa Xaa Tyr Xaa Xaa Xaa Xaa XaaTyr Xaa Xaa Xaa Xaa Tyr 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 127 21 PRTArtificial Sequence Complement C1s inhibitor 127 Pro Asn Xaa Xaa Tyr XaaXaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Tyr 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20128 21 PRT Artificial Sequence Formula of a C1s exosite binding moiety128 Ala Asn Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Tyr 1 510 15 Xaa Xaa Xaa Xaa Xaa 20 129 20 PRT Artificial Sequence Formula of acomplement C1s inhibitor 129 Pro Asn Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa TyrXaa Xaa Xaa Xaa Tyr 1 5 10 15 Xaa Xaa Xaa Xaa 20 130 19 PRT ArtificialSequence Formula of a complement C1s inhibitor. 130 Pro Asn Glu Glu TyrXaa Xaa Xaa Glu Tyr Xaa Xaa Xaa Glu Tyr Xaa 1 5 10 15 Xaa Xaa Glu 131 7PRT Artificial Sequence Peptide inker 131 Ala Leu Xaa Xaa Xaa Xaa Xaa 15 132 7 PRT Artificial Sequence Peptide inker 132 Ala Leu Xaa Xaa XaaXaa Xaa 1 5 133 7 PRT Artificial Sequence Peptide inker 133 Xaa Xaa XaaXaa Xaa Xaa Xaa 1 5 134 7 PRT Artificial Sequence Peptide inker 134 XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 135 7 PRT Artificial Sequence Peptide inker135 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 136 7 PRT Artificial SequencePeptide inker 136 Ala Leu Xaa Xaa Xaa Xaa Cys 1 5 137 7 PRT ArtificialSequence Peptide inker 137 Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 138 7 PRTArtificial Sequence Peptide inker 138 Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5139 7 PRT Artificial Sequence Peptide inker 139 Ala Leu Xaa Xaa Xaa XaaCys 1 5 140 17 PRT Artificial Sequence C1s catalytic site directedmoiety 140 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa LeuGln 1 5 10 15 Arg

We claim:
 1. A polypeptide that inhibits complement C1s, wherein thepolypeptide is characterized by the formula:“P-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”where amino acid residues in square brackets indicate acceptable aminoacids, numbers in parentheses indicate the number of amino acidresidues, “X₁” represents Phe-(p-CH₂)SO₃H, “X₂” represents sulfatedtyrosine, and “X₃” represents 2-sulfotyrosine (SEQ ID NO: 127).
 2. Thepolypeptide of claim 1, wherein the polypeptide is characterized by theformula: “P-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE]”(SEQ ID NO:129).
 3. The polypeptide of claim 2, wherein the polypeptideis characterized by the formula:“P-N-E-E-[YX₁X₂X₃]-E-[YX₁X₂X₃]-E-[YX₁X₂X₃]-E” (SEQ ID NO: 130).
 4. Thepolypeptide of claim 3, wherein the polypeptide consists of the aminoacid sequence: “PNEEY EYEYE” (SEQ ID NO:125).


5. A polypeptide that inhibits complement C1s, wherein the polypeptidecomprises an amino acid sequence that is characterized by the formula:“[AP]-N-[DE](2)-[X₁X₂X₃]-[DE](2)-[X₁X₂X₃]-[DE]-[X₁X₂X₃]-[DE](1,2)” whereamino acid residues in square brackets indicate acceptable amino acids,numbers in parentheses indicate the number of amino acid residues, “X₁”represents Phe-(p-CH₂)SO₃H, “X₂” represents sulfated tyrosine, and “X₃”represents 2-sulfotyrosine (SEQ ID NO: 126).
 6. A peptide or polypeptidethat inhibits complement C1s, wherein the peptide or polypeptidecomprises the amino acid sequence “CRLGC” (amino acid residues 64 to 68of SEQ ID NO: 1), wherein the peptide or polypeptide consists of five tothirty amino acid residues.
 7. The peptide or polypeptide of claim 6,wherein the polypeptide consists of the amino acid sequence: “GCDGFKCRLGCTYGFKTDKK GCEAFCTCNT” (SEQ ID NO:53).


8. The peptide of claim 6, wherein the peptide consists of the aminoacid sequence: “CRLGC.”
 9. A complement C1s inhibitor, wherein theinhibitor consists of: (a) a C1s catalytic site-directed moiety (CCSDM),which is selected from the group consisting of: (i)CH₃-Lys(Cbo)-Gly-Arg-pNA-AcOH, where “Cbo” represents benzyloxycarbonyl;(ii) CH₃-Lys(Cbo)-Gly-Arg; (iii) H-D-Val-Ser-Arg-pNA.HCl; (iv)H-D-Val-Ser-Arg; (v) Leu-Xaa-Arg, where “Xaa” represents alanine,glutamine, or glycine; (vi) LQRALEILPN RVTIKANRPF LVFI (SEQ ID NO:118),(vii) serine protease inhibitor; (viii) heterocyclic protease inhibitor;(ix) transition state analogue; (x) benzamidine; (xi) X-C1-C2-A-Y, whereC1 is a derivative of Arg, Lys, or Om, characterized by a reducedcarboxylate moiety or a carboxylate moiety that is displaced from theα-carbon by a chemical structure characterized by a backbone chain offrom 1 to 10 atoms, C2 is a non-cleavable bond, “X” is hydrogen or acontinuation of the peptide backbone, “A” is a backbone chain, and “Y”is a bond; (xii) CDGFK CRLGC TYGFK TDKKG CEAFC TCNT (SEQ ID NO:121); and(xiii) X-C-X(8-12)-L-Q-R, where “X” represents glycine, serine, orthreonine, and numbers in parentheses indicate the number of amino acidresidues (SEQ ID NO:140); (b) a linker moiety that is eithercharacterized by a backbone chain having a calculated length of between14 Å and 20 Å, or that is a polypeptide, which has the amino acidsequence of KETAC VNIWC TDPYK CNPES GRCED (SEQ ID NO:123);

 and (c) a C1s exosite binding moiety (CEBM), which is selected from thegroup consisting of: (i) a polypeptide characterized by the formula:“[AP]-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”where amino acid residues in square brackets indicate acceptable aminoacids, numbers in parentheses indicate the number of amino acidresidues, “X₁” represents Phe-(p-CH2)SO₃H, “X₂” represents sulfatedtyrosine, and “X₃” represents 2-sulfotyrosine (SEQ ID NO:126); and (ii)NEDYEDYEYD (SEQ ID NO:119);

wherein the C1s catalytic site-directed moiety is bound to the linkermoiety, the linker moiety is bound to the C1s exosite binding moiety.10. The inhibitor of claim 9, wherein the serine protease inhibitor isselected from the group consisting of phenylmethylsulfonylfluoride,diisopropylflouorophosphate, tosylprolylchloromethylketone, andtosyllysl chloromethylketone.
 11. The inhibitor of claim 9, wherein theheterocyclic protease inhibitor is an isocoumarin.
 12. The inhibitor ofclaim 9, wherein the transition state analogue is difluoroketomethylene.13. The inhibitor of claim 9, wherein a moiety having the formula“X-C1-C2-A-Y” includes a C1 component selected from the group consistingof β-homoarginine, an arginine containing a reduced carboxylate moiety,and β-homoornithine.
 14. The inhibitor of claim 13, wherein the argininecontaining a reduced carboxylate moiety is Argψ[CH₂NH].
 15. Theinhibitor of claim 13, wherein the linker is selected from the groupconsisting of: (i) A-L-[ED]-[ED]-X(1-3) (SEQ ID NO:131), (ii)A-L-X(1-3)-[ED]-[ED](SEQ ID NO:132), (iii) A-L-[ED]-[ED](SEQ ID NO:122),(iv) X(2-5)-[ED]-[ED](SEQ ID NO:134), (v) A-L-[ED]-[ED]-X(1-2)-C (SEQ IDNO:136), (vi) A-L-[ED]-[ED]-C (SEQ ID NO:124), (vii) X(1-4)-[ED]-[ED]-C(SEQ ID NO:138), (viii) A-L-X(1-2)-[ED]-[ED]-C (SEQ ID NO:139), (ix)X(4-7) (SEQ ID NO:133), (x) X(5-7) (SEQ ID NO:135), and (xi) X(3-6)-C(SEQ ID NO:137),

where amino acid residues in square brackets indicate acceptable aminoacids, numbers in parentheses indicate the number of amino acidresidues, and “X” represents any of glycine, serine, or threonine.
 16. Acomplement C1s inhibitor, wherein the inhibitor consists of: (a) a C1scatalytic site-directed moiety (CCSDM), which is selected (i) GCDGFKCRLGCTYGFKTDKK GCEAFCTCNT (SEQ ID NO:53);

 from the group consisting of: and (ii) CRLGC (amino acid residues 64 to68 of SEQ ID NO:1); (b) a linker moiety characterized by a backbonechain having a calculated length of between 14 Å and 20 Å; and (c) a C1sexosite binding moiety (CEBM), which is a polypeptide characterized bythe formula:“A-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”where amino acid residues in square brackets indicate acceptable aminoacids, numbers in parentheses indicate the number of amino acidresidues, “X₁” represents Phe-(p-CH2)SO₃H, “X₂” represents sulfatedtyrosine, “X₃” represents 2-sulfotyrosine (SEQ ID NO: 128); wherein theC1s catalytic site-directed moiety is bound to the linker moiety, thelinker moiety is bound to the C1s exosite binding moiety.
 17. Thecomplement C1s inhibitor of any one of claims 9 or 16, wherein theinhibitor is characterized by the formula: “CCSDM-Linker-CEBM.” 18.composition, comprising a carrier, and a peptide or a polypeptide of anyone of claims 1, 5, or
 6. 19. A composition, comprising a carrier, andthe complement C1s inhibitor of any one of claims 9 or
 16. 20. A methodof inhibiting complement C1s inhibitor, comprising administering thecomposition of claim 18 to complement C1 s.
 21. A method of inhibitingcomplement C1s inhibitor, comprising administering the composition ofclaim 19 to complement C1s.
 22. The method of claim 20, wherein thecomposition is administered to a mammalian subject.
 23. The method ofclaim 21, wherein the composition is administered to a mammaliansubject.